Bispecific antibody against bcma and cd3 and an immunological drug for combined use in treating multiple myeloma

ABSTRACT

The invention relates to a bispecific antibody specifically binding to human B cell maturation antigen (BCMA) and to human CD3ε (CD3) together with an immunotherapeutic drug for combined use in treating multiple myeloma.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation application of U.S.application Ser. No. 16/346,771, filed May 1, 2019, which is a 35 U.S.C.§ 371 national phase application of International Application No.PCT/EP2017/078109, filed Nov. 2, 2017, and published under PCT Article21(2) in English, which designated the U.S., and claims the benefit ofpriority from European Application No. EP 16196874.8, filed Nov. 2,2016, and each of which prior applications are incorporated by referenceherein into this application in their entirety including all tables,figures and claims.

SEQUENCE LISTING

The instant application contains a Sequence Listing which is submittedin ASCII format via EFS-Web and is hereby incorporated by reference inits entirety. Said ASCII copy, created on Jun. 24, 2021, is named298068-00362_Sequence_Listing.txt and is 68,085 bytes in size.

FIELD OF THE INVENTION

The present invention relates to a bispecific antibody specificallybinding to human B cell maturation antigen (BCMA) and to human CD3ε(CD3) together with an immunotherapeutic drug for combined use intreating multiple myeloma.

BACKGROUND OF THE INVENTION

Human B cell maturation antigen, also known as BCMA; TR17_HUMAN,TNFRSF17 (UniProt Q02223), is a member of the tumor necrosis receptorsuperfamily that is preferentially expressed in differentiated plasmacells (Laabi et al. 1992; Madry et al. 1998). BCMA is a non-glycosylatedtype III transmembrane protein, which is involved in B cell maturation,growth and survival. BCMA is a receptor for two ligands of the TNFsuperfamily: APRIL (a proliferation-inducing ligand), the high-affinityligand to BCMA and the B cell activation factor BAFF, the low-affinityligand to BCMA (THANK, BlyS, B lymphocyte stimulator, TALL-1 and zTNF4).APRIL and BAFF show structural similarity and overlapping yet distinctreceptor binding specificity. The negative regulator TACI also binds toboth BAFF and APRIL. The coordinate binding of APRIL and BAFF to BCMAand/or TACI activates transcription factor NF-κB and increases theexpression of pro-survival Bcl-2 family members (e.g. Bcl-2, Bcl-xL,Bcl-w, Mcl-1, A1) and the downregulation of pro-apoptotic factors (e.g.Bid, Bad, Bik, Bim, etc.), thus inhibiting apoptosis and promotingsurvival. This combined action promotes B cell differentiation,proliferation, survival and antibody production (as reviewed in RickertR C et al., Immunol Rev (2011) 244 (1): 115-133).

Antibodies against BCMA are described e.g. in Gras M-P. et al. IntImmunol. 7 (1995) 1093-1106, WO200124811, WO200124812, WO2010104949 andWO2012163805. Antibodies against BCMA and their use for the treatment oflymphomas and multiple myeloma are mentioned e.g. in WO2002066516 andWO2010104949. WO2013154760 and WO2015052538 relate to chimeric antigenreceptors (CAR) comprising a BCMA recognition moiety and a T-cellactivation moiety. Ryan, M C et al., Mol. Cancer Ther. 6 (2007)3009-3018 relate to anti BCMA antibodies with ligand blocking activitythat could promote cytotoxicity of multiple myeloma (MM) cell lines asnaked antibodies or as antibody-drug conjugates. Ryan showed that SG1,an inhibitory BCMA antibody, blocks APRIL-dependent activation ofnuclear factor-κB in a dose-dependent manner in vitro. Ryan alsomentioned antibody SG2 which inhibited APRIL binding to BCMA notsignificantly.

A wide variety of recombinant bispecific antibody formats have beendeveloped in the recent past, e.g. by fusion of, e.g. an IgG antibodyformat and single chain domains (see e.g. Kontermann R E, mAbs 4:2,(2012) 1-16). Bispecific antibodies wherein the variable domains VL andVH or the constant domains CL and CH1 are replaced by each other aredescribed in WO2009080251 and WO2009080252.

An approach to circumvent the problem of mispaired byproducts, which isknown as ‘knobs-into-holes’, aims at forcing the pairing of twodifferent antibody heavy chains by introducing mutations into the CH3domains to modify the contact interface. On one chain bulky amino acidswere replaced by amino acids with short side chains to create a ‘hole’.Conversely, amino acids with large side chains were introduced into theother CH3 domain, to create a ‘knob’. By coexpressing these two heavychains (and two identical light chains, which have to be appropriate forboth heavy chains), high yields of heterodimer formation (‘knob-hole’)versus homodimer formation (‘hole-hole’ or ‘knob-knob’) was observed(Ridgway J B, Presta L G, Carter P. Protein Eng. 9, 617-621 (1996); andWO1996027011). The percentage of heterodimer could be further increasedby remodeling the interaction surfaces of the two CH3 domains using aphage display approach and the introduction of a disulfide bridge tostabilize the heterodimers (Merchant A. M, et al, Nature Biotech 16(1998) 677-681; Atwell S, Ridgway J B, Wells J A, Carter P., J Mol. Biol270 (1997) 26-35). New approaches for the knobs-into-holes technologyare described in e.g. in EP 1870459A1. Although this format appears veryattractive, no data describing progression towards the clinic arecurrently available. One important constraint of this strategy is thatthe light chains of the two parent antibodies have to be identical toprevent mispairing and formation of inactive molecules. Thus thistechnique is not appropriate for easily developing recombinant,bispecific antibodies against two targets starting from two antibodiesagainst the first and the second target, as either the heavy chains ofthese antibodies and/or the identical light chains have to be optimized.Xie, Z., et al, J Immunol. Methods 286 (2005) 95-101 refers to a formatof bispecific antibody using scFvs in combination with knobs-into-holestechnology for the FC part.

The TCR/CD3 complex of T-lymphocytes consists of either a TCR alpha(a)/beta 03) or TCR gamma (γ)/delta (δ) heterodimer coexpressed at thecell surface with the invariant subunits of CD3 labeled gamma (γ), delta(δ), epsilon (ε), zeta (ζ), and eta (η). Human CD3ε is described underUniProt P07766 (CD3E_HUMAN).

An anti CD3ε antibody described in the state of the art is SP34 (Yang SJ, The Journal of Immunology (1986) 137; 1097-1100). SP34 reacts withboth primate and human CD3. SP34 is available from Pharmingen. A furtheranti CD3 antibody described in the state of the art is UCHT-1 (seeWO2000041474). A further anti CD3 antibody described in the state of theart is BC-3 (Fred Hutchinson Cancer Research Institute; used in PhaseI/II trials of GvHD, Anasetti et al., Transplantation 54: 844 (1992)).SP34 differs from UCHT-1 and BC-3 in that SP-34 recognizes an epitopepresent on solely the ε chain of CD3 (see Salmeron et al., (1991) J.Immunol. 147: 3047) whereas UCHT-1 and BC-3 recognize an epitopecontributed by both the ε and γ chains. Further anti-CD3 antibodies aredescribed in WO2008119565, WO2008119566, WO2008119567, WO2010037836,WO2010037837, WO2010037838, and U.S. Pat. No. 8,236,308 (WO2007042261).CDRs, VH and VL sequences of a further anti-CD3 antibody are shown inSEQ ID NO:7 and 8.

Bispecific antibodies against CD3 and BCMA are mentioned inWO2007117600, WO2009132058, WO2012066058, and WO2012143498. CARcompounds of antibodies against BCMA are mentioned in WO2013154760,WO2013154760, and WO2014140248.

Cell-mediated effector functions of monoclonal antibodies (like antibodydependent cellular cytotoxicity (ADCC)) can be enhanced by engineeringtheir oligosaccharide composition at Asn297 as described in Umaña, P.,et al., Nature Biotechnol. 17 (1999) 176-180; and U.S. Pat. No.6,602,684. WO1999054342, WO2004065540, WO2007031875, and WO2007039818,Hristodorov D, Fischer R, Linden L., Mol Biotechnol. 2012 Oct. 25.(Epub) also relate to the glycosylation engineering of antibodies toenhance Fc-mediated cellular cytotoxicity.

Also several amino acid residues in the hinge region and the CH2 domaininfluence cell-mediated effector functions of monoclonal antibodies(Eur. J. Immunol., 23, 1098 (1993), Immunology, 86, 319 (1995), ChemicalImmunology, 65, 88 (1997)] Chemical Immunology, 65, 88 (1997)].Therefore modification of such amino acids can enhance cell-mediatedeffector functions. Such antibody modifications to increasecell-mediated effector functions are mentioned in EP1931709, WO200042072and comprise in the Fc part substitutions at amino acid position(s) 234,235, 236, 239, 267, 268, 293, 295, 324, 327, 328, 330, and 332.

Further antibody modifications to increase cell-mediated effectorfunctions are mentioned in EP1697415 and comprise amino acid replacementof EU amino acid positions 277, 289, 306, 344, or 378 with a chargedamino acid, a polar amino acid, or a nonpolar amino acid.

Antibody formats and formats of bispecific and multispecific antibodiesare also pepbodies (WO200244215), Novel Antigen Receptor (“NAR”)(WO2003014161), diabody-diabody dimers “TandAbs” (WO2003048209),polyalkylene oxide-modified scFv (U.S. Pat. No. 7,150,872), humanizedrabbit antibodies (WO2005016950), synthetic immunoglobulin domains(WO2006072620), covalent diabodies (WO2006113665), flexibodies(WO2003025018), domain antibodies, dAb (WO2004058822), vaccibody(WO2004076489), antibodies with new world primate framework(WO2007019620), antibody-drug conjugate with cleavable linkers(WO2009117531), IgG4 antibodies with hinge region removed(WO2010063785), bispecific antibodies with IgG4 like CH3 domains(WO2008119353), camelid Antibodies (U.S. Pat. No. 6,838,254), nanobodies(U.S. Pat. No. 7,655,759), CAT diabodies (U.S. Pat. No. 5,837,242),bispecific (scFv)₂ directed against target antigen and CD3 (U.S. Pat.No. 7,235,641), sIgA plAntibodies (U.S. Pat. No. 6,303,341), minibodies(U.S. Pat. No. 5,837,821), IgNAR (US2009148438), antibodies withmodified hinge and Fc regions (US2008227958, US20080181890),trifunctional antibodies (U.S. Pat. No. 5,273,743), triomabs (U.S. Pat.No. 6,551,592), troybodies (U.S. Pat. No. 6,294,654).

WO2014122143 disclose anti-human BCMA antibodies characterized in thatthe binding of said antibody is not reduced by 100 ng/ml APRIL for morethan 20% measured in an ELISA assay as OD at 405 nm compared to thebinding of said antibody to human BCMA without APRIL, said antibody doesnot alter APRIL-dependent NF-κB activation for more than 20%, ascompared to APRIL alone, and said antibody does not alter NF-κBactivation without APRIL for more than 20%, as compared without saidantibody. WO2014122144 discloses bispecific antibodies specificallybinding to the two targets human CD3ε and human BCMA, comprisinganti-human BCMA antibodies of WO2014122143. An anti-human BCMA antibodywith unique properties, especially in regard to its therapeutic use as abispecific T cell binder, is antibody 83A10, characterized by comprisingas CDR regions CDR1H of SEQ ID NO:15, CDR2H of SEQ ID NO16, CDR3H of SEQID NO:17, CDR1L of SEQ ID NO:18, CDR3L of SEQ ID NO:19, and CDR3L of SEQID NO:20, disclosed also in WO2014122143 and WO2014122144.

Thalidomide compounds are2-(2,6-dioxopiperidin-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione andderivatives thereof like lenalidomide, pomalidomide, CC122 (CAS RegistryNumber 1398053-45-6) or CC-220 (CAS Registry Number 1323403-33-3). Theuse of thalidomides for the treatment of multiple myeloma is describedin Hideshima T. et al., Blood 96 (2000), 2943-2950.

Anti-CD38 antibodies are e.g. daratumumab (US20150246123), isatuximab(U.S. Pat. No. 8,877,899), MOR202 (WO 2012041800), and AB19 and AB79(U.S. Pat. No. 8,362,211). Anti-CD38 antibodies are also mentioned inWO2006099875, WO2011154453, WO2014068114, and WO2007042309. The use ofan anti-CD38 monoclonal antibody in the treatment of multiple myeloma ise.g. discussed by Lokhorst H M; N Engl J Med (2015) 373 1207-19.

Anti-PD-1 antibodies are e.g. pembrolizumab (Keytruda®, MK-3475),nivolumab, pidilizumab, lambrolizumab, MEDI-0680, PDR001, and REGN2810.Anti-PD-1 antibodies are described e.g. in WO200815671, WO2013173223,WO2015026634, U.S. Pat. Nos. 7,521,051, 8,008,449, 8,354,509,WO2009114335, WO2015026634, WO2008156712, WO2015026634, WO2003099196,WO2009101611, WO2010/027423, WO2010/027827, WO2010/027828,WO2008/156712, and WO2008/156712.

Anti-PD-L1 antibodies are e.g. atezolizumab, MDX-1105, durvalumab andavelumab. Anti-PD-L1 antibodies are e.g. described in WO2015026634,WO2013/019906, WO2010077634, U.S. Pat. No. 8,383,796, WO2010077634,WO2007005874, and WO2016007235.

WO2012066058 mentions in general terms a bispecific agent against BCMAand CD3 also in combination with one or more additional therapeuticagents. A lot of such agents are listed and among others, alsothalidomide derivatives like lenalidomide are mentioned. WO2012143498mentions also in general terms an anti-BCMA antibody therapy for use inthe treatment or amelioration of a multiple myeloma and an anti-CD20antibody and/or an anti-CD38 antibody and/or an anti-CS1 antibodytherapy. WO2016087531 discloses the use of a bispecific antibody againstBCMA and CD3 together with a T-cell proliferative therapy” refers to atherapeutic treatment or a biological treatment which induces theproliferation or expansion of T cells such as e.g. checkpoint inhibitors(e.g. anti-PD-1, anti-PD-L1). WO2016014565 relates to chimeric antigenreceptor (CAR) specific to BCMA and in general terms an antibody orantibody fragment that binds to PD-1, PD-L1, PD-L2 or CTLA4.

The bispecific antibodies and the respective anti-BCMA antibodies of thepresent invention are disclosed in EP15179549.9 and PCT/EP2016/068549,now pending, which are hereby incorporated in whole by reference.

SUMMARY OF THE INVENTION

The invention comprises a bispecific antibody specifically binding tohuman B cell maturation antigen (BCMA) and to human CD3ε (CD3) togetherwith an immunotherapeutic drug selected from the group consisting ofthalidomide and an immunotherapeutic derivative thereof, an anti-CD38antibody, an anti-PD-1 antibody and an anti-PD-L1 antibody, for combineduse in treating multiple myeloma.

The invention comprises in one embodiment

a) a bispecific antibody comprising a first binding part specificallybinding to human B cell maturation antigen (BCMA) and a second bindingpart specifically binding to human CD3ε (CD3) and

b) an immunotherapeutic drug selected from the group consisting ofthalidomide and an immunotherapeutic derivative thereof, an anti-CD38antibody, an anti-PD-1 antibody and an anti-antibody and an anti-PD-L1antibody, for combined use in treating multiple myeloma,

characterized in that said first binding part comprises a VH regioncomprising a CDR1H region of SEQ ID NO:21, a CDR2H region of SEQ IDNO:22 and a CDR3H region of SEQ ID NO:17 and a VL region comprising aCDR3L region of SEQ ID NO:20 and a CDR1L and CDR2L region combinationselected from the group of

i) CDR1L region of SEQ ID NO:23 and CDR2L region of SEQ ID NO:24,

ii) CDR1L region of SEQ ID NO:25 and CDR2L region of SEQ ID NO:26, or

iii) CDR1L region of SEQ ID NO:27 and CDR2L region of SEQ ID NO:28.

The invention comprises in one embodiment a method of treating multiplemyeloma, characterized in administering to a patient in need of suchtreatment

a) a bispecific antibody comprising a first binding part specificallybinding to human B cell maturation antigen (BCMA) and a second bindingpart specifically binding to human CD3ε (CD3) and

b) an immunotherapeutic drug selected form the group consisting ofthalidomide and an immunotherapeutic derivative thereof, an anti-CD38antibody, an anti-PD-1 antibody and an anti-PD-L1 antibody,

characterized in that said first binding part comprises a VH regioncomprising a CDR1H region of SEQ ID NO:21, a CDR2H region of SEQ IDNO:22 and a CDR3H region of SEQ ID NO:17 and a VL region comprising aCDR3L region of SEQ ID NO:20 and a CDR1L and CDR2L region combinationselected from the group of

i) CDR1L region of SEQ ID NO:23 and CDR2L region of SEQ ID NO:24,

ii) CDR1L region of SEQ ID NO:25 and CDR2L region of SEQ ID NO:26, or

iii) CDR1L region of SEQ ID NO:27 and CDR2L region of SEQ ID NO:28.

The bispecific antibody and the immunotherapeutic drug are used in atherapeutically effective amount.

The invention comprises in one embodiment a therapeutic combination forachieving multiple myeloma cell lysis in a patient suffering frommultiple myeloma disease, characterized in comprising a

a) a bispecific antibody comprising a first binding part specificallybinding to human B cell maturation antigen (BCMA) and a second bindingpart specifically binding to human CD3ε (CD3) and

b) an immunotherapeutic drug selected form the group consisting ofthalidomide and an immunotherapeutic derivative thereof, an anti-CD38antibody, an anti-PD-1 antibody and an anti-PD-L1 antibody,

characterized in that said first binding part comprises a VH regioncomprising a CDR1H region of SEQ ID NO:21, a CDR2H region of SEQ IDNO:22 and a CDR3H region of SEQ ID NO:17 and a VL region comprising aCDR3L region of SEQ ID NO:20 and a CDR1L and CDR2L region combinationselected from the group of

i) CDR1L region of SEQ ID NO:23 and CDR2L region of SEQ ID NO:24,

ii) CDR1L region of SEQ ID NO:25 and CDR2L region of SEQ ID NO:26, or

iii) CDR1L region of SEQ ID NO:27 and CDR2L region of SEQ ID NO:28.

The bispecific antibody and the immunotherapeutic drug are used in atherapeutically effective amount.

The invention comprises in one embodiment an article of manufacture,characterized in comprising a) a bispecific antibody comprising a firstbinding part specifically binding to human B cell maturation antigen(BCMA) and a second binding part specifically binding to human CD3ε(CD3), characterized in that said first binding part comprises a VHregion comprising a CDR1H region of SEQ ID NO:21, a CDR2H region of SEQID NO:22 and a CDR3H region of SEQ ID NO:17 and a VL region comprising aCDR3L region of SEQ ID NO:20 and a CDR1L and CDR2L region combinationselected from the group of

i) CDR1L region of SEQ ID NO:23 and CDR2L region of SEQ ID NO:24,

ii) CDR1L region of SEQ ID NO:25 and CDR2L region of SEQ ID NO:26, or

iii) CDR1L region of SEQ ID NO:27 and CDR2L region of SEQ ID NO:28. in apharmaceutically acceptable carrier,

b) an immunotherapeutic drug selected form the group consisting ofthalidomide and an immunotherapeutic derivative thereof, an anti-CD38antibody, an anti-PD-1 antibody and an anti-PD-L1 antibody,

c) a pharmaceutically acceptable carrier and instructions foradministering said bispecific antibody and said immunotherapeutic drugin combination to a subject in need of a treatment for multiple myeloma.

The bispecific antibody and the immunotherapeutic drug are used in atherapeutically effective amount.

The invention comprises in one embodiment a method for manufacturing amedicament, characterized in using

a)) a bispecific antibody comprising a first binding part specificallybinding to human B cell maturation antigen (BCMA) and a second bindingpart specifically binding to human CD3ε (CD3), characterized in thatsaid first binding part comprises a VH region comprising a CDR1H regionof SEQ ID NO:21, a CDR2H region of SEQ ID NO:22 and a CDR3H region ofSEQ ID NO:17 and a VL region comprising a CDR3L region of SEQ ID NO:20and a CDR1L and CDR2L region combination selected from the group of

i) CDR1L region of SEQ ID NO:23 and CDR2L region of SEQ ID NO:24,

ii) CDR1L region of SEQ ID NO:25 and CDR2L region of SEQ ID NO:26, or

iii) CDR1L region of SEQ ID NO:27 and CDR2L region of SEQ ID NO:28,

b) an immunotherapeutic drug selected form the group consisting ofthalidomide and an immunotherapeutic derivative thereof, an anti-CD38antibody, an anti-PD-1 antibody and an anti-PD-L1 antibody,

c) combining said bispecific antibody and said immunotherapeutic drug ina pharmaceutically acceptable carrier.

The bispecific antibody and the immunotherapeutic drug are used in atherapeutically effective amount.

The bispecific antibodies and the immunotherapeutic drugs, describedherein, are for use

-   -   a) in the combined use in treating multiple myeloma according to        the invention,    -   b) in the method of treating multiple myeloma according to the        invention,    -   c) in the therapeutic combination for achieving multiple myeloma        cell lysis in a patient suffering from multiple myeloma disease        according to the invention,    -   d) in the article of manufacture according to the invention, and    -   e) in the method for manufacturing a medicament according to the        invention.

The invention comprises in one embodiment that said immunotherapeuticdrug is an anti-CD38 antibody selected from the group consisting ofdaratumumab, isatuximab (SAR650984), MOR202, Ab79 (Takeda) and Ab19(Takeda).

The invention comprises in one embodiment that said immunotherapeuticdrug is a thalidomide compound selected from the group consisting ofthalidomide, lenalidomide, CC-122, CC-220, and pomalidomide.

The invention comprises in one embodiment that said immunotherapeuticdrug is an anti-PD1 antibody, selected from the group consisting ofpembrolizumab, pidilizumab, nivolumab, MEDI-0680, PDR001, REGN2810,lambrolizumab, MDX-1106, BGB-108, h409A11, h409A16 and h409A17.

The invention comprises in one embodiment that said immunotherapeuticdrug is an anti-PD-L1 antibody, selected from the group consisting ofavelumab, durvalumab, atezolizumab, and MDX-1105.

The invention comprises in one embodiment that said immunotherapeuticdrug is lenalidomide and the first binding part is characterized incomprising a VH region comprising a CDR1H region of SEQ ID NO:21, aCDR2H region of SEQ ID NO:22 and a CDR3H region of SEQ ID NO:17 and a VLregion comprising a CDR3L region of SEQ ID NO:20 and a CDR1L and CDR2Lregion combination selected from the group of

-   -   a) CDR1L region of SEQ ID NO:25 and CDR2L region of SEQ ID        NO:26, or    -   b) CDR1L region of SEQ ID NO:27 and CDR2L region of SEQ ID        NO:28.

The invention comprises in one embodiment that said immunotherapeuticdrug is CC-122 or CC-220 and the first binding part is characterized incomprising a VH region comprising a CDR1H region of SEQ ID NO:21, aCDR2H region of SEQ ID NO:22 and a CDR3H region of SEQ ID NO:17 and a VLregion comprising a CDR3L region of SEQ ID NO:20 and a CDR1L and CDR2Lregion combination selected from the group of

a) CDR1L region of SEQ ID NO:25 and CDR2L region of SEQ ID NO:26, or

b) CDR1L region of SEQ ID NO:27 and CDR2L region of SEQ ID NO:28.

The invention comprises in one embodiment that said immunotherapeuticdrug is daratumumab and the first binding part is characterized incomprising a VH region comprising a CDR1H region of SEQ ID NO:21, aCDR2H region of SEQ ID NO:22 and a CDR3H region of SEQ ID NO:17 and a VLregion comprising a CDR3L region of SEQ ID NO:20 and a CDR1L and CDR2Lregion combination selected from the group of

-   -   a) CDR1L region of SEQ ID NO:25 and CDR2L region of SEQ ID        NO:26, or    -   b) CDR1L region of SEQ ID NO:27 and CDR2L region of SEQ ID        NO:28.

The invention comprises in one embodiment that said immunotherapeuticdrug is pembrolizumab and the first binding part is characterized incomprising a VH region comprising a CDR1H region of SEQ ID NO:21, aCDR2H region of SEQ ID NO:22 and a CDR3H region of SEQ ID NO:17 and a VLregion comprising a CDR3L region of SEQ ID NO:20 and a CDR1L and CDR2Lregion combination selected from the group of

-   -   a) CDR1L region of SEQ ID NO:25 and CDR2L region of SEQ ID        NO:26, or    -   b) CDR1L region of SEQ ID NO:27 and CDR2L region of SEQ ID        NO:28.

The invention comprises in one embodiment that said immunotherapeuticdrug is lenalidomide and the first binding part is characterized incomprising a VH region of SEQ ID NO:10 and a VL region of SEQ ID NO:13or a VH region of SEQ ID NO:10 and a VL region of SEQ ID NO:14.

The invention comprises in one embodiment that said immunotherapeuticdrug is CC-122 or CC-220 and the first binding part is characterized incomprising a VH region of SEQ ID NO:10 and a VL region of SEQ ID NO:13or a VH region of SEQ ID NO:10 and a VL region of SEQ ID NO:14

The invention comprises in one embodiment that said immunotherapeuticdrug is daratumumab and the first binding part is characterized incomprising a VH region of SEQ ID NO:10 and a VL region of SEQ ID NO:13or a VH region of SEQ ID NO:10 and a VL region of SEQ ID NO:14.

The invention comprises in one embodiment that said immunotherapeuticdrug is pembrolizumab and the first binding part is characterized incomprising a VH region of SEQ ID NO:10 and a VL region of SEQ ID NO:13or a VH region of SEQ ID NO:10 and a VL region of SEQ ID NO:14.

The first binding part according to the invention comprises as CDR3H andCDR3L regions the same CDR regions as antibody 83A10 (for antibody 83A10see table 1A and B later in the text).

The first binding part according to the invention comprises in oneembodiment as CDR3H and CDR3L regions the same CDR regions as antibody83A10, but show especially potent and efficient advantages in comparisonto antibody 83A10 for killing of MM cells in patient bone marrowaspirates.

In one embodiment of the invention the first binding part ischaracterized in comprising a CDR3H region of SEQ ID NO:17 and a CDR3Lregion of SEQ ID NO:20 and a CDR1H, CDR2H, CDR1L, and CDR2L regioncombination selected from the group of

a) CDR1H region of SEQ ID NO:21 and CDR2H region of SEQ ID NO:22, CDR1Lregion of SEQ ID NO:23, and CDR2L region of SEQ ID NO:24,

b) CDR1H region of SEQ ID NO:21 and CDR2H region of SEQ ID NO:22, CDR1Lregion of SEQ ID NO:25, and CDR2L region of SEQ ID NO:26,

c) CDR1H region of SEQ ID NO:21 and CDR2H region of SEQ ID NO:22, CDR1Lregion of SEQ ID NO:27, and CDR2L region of SEQ ID NO:28,

d) CDR1H region of SEQ ID NO:29 and CDR2H region of SEQ ID NO:30, CDR1Lregion of SEQ ID NO:31, and CDR2L region of SEQ ID NO:32,

e) CDR1H region of SEQ ID NO:34 and CDR2H region of SEQ ID NO:35, CDR1Lregion of SEQ ID NO:31, and CDR2L region of SEQ ID NO:32, and

f) CDR1H region of SEQ ID NO:36 and CDR2H region of SEQ ID NO:37, CDR1Lregion of SEQ ID NO:31, and CDR2L region of SEQ ID NO:32.

In one embodiment of the invention the first binding part ischaracterized in comprising a VH region comprising a CDR1H region of SEQID NO:21, a CDR2H region of SEQ ID NO:22 and a CDR3H region of SEQ IDNO:17 and a VL region comprising a CDR3L region of SEQ ID NO:20 and aCDR1L and CDR2L region combination selected from the group of

a) CDR1L region of SEQ ID NO:23 and CDR2L region of SEQ ID NO:24,

b) CDR1L region of SEQ ID NO:25 and CDR2L region of SEQ ID NO:26, or

c) CDR1L region of SEQ ID NO:27 and CDR2L region of SEQ ID NO:28.

In one embodiment of the invention the first binding part ischaracterized in comprising a VL region selected from the groupconsisting of VL regions of SEQ ID NO:12, 13, and 14 wherein amino acid49 is selected from the group of amino acids tyrosine (Y), glutamic acid(E), serine (S), and histidine (H). In one embodiment amino acid 49 is Ewithin SEQ ID NO:12, S within SEQ ID NO:13 or H within SEQ ID NO:14.

In one embodiment of the invention the first binding part ischaracterized in comprising a VL region selected from the groupconsisting of VL regions of SEQ ID NO:12, 13, and 14 wherein amino acid74 is threonine (T) or alanine (A). In one embodiment amino acid 74 is Awithin SEQ ID NO:14.

In one embodiment of the invention the first binding part comprise asCDR3H, CDR1L, CDR2L, and CDR3L regions the same CDR regions as antibody83A10. The invention comprises a monoclonal antibody specificallybinding to BCMA, characterized in comprising a VH region comprising aCDR3H region of SEQ ID NO:17 and a VL region comprising a CDR1L regionof SEQ ID NO:31, a CDR2L region of SEQ ID NO:32 and a CDR3L region ofSEQ ID NO:20 and a CDR1L and CDR2L region combination selected from thegroup of

a) CDR1H region of SEQ ID NO:29 and CDR2H region of SEQ ID NO:30,

b) CDR1H region of SEQ ID NO:34 and CDR2H region of SEQ ID NO:35, or

c) CDR1H region of SEQ ID NO:36 and CDR2H region of SEQ ID NO:37.

In one embodiment of the invention the first binding part ischaracterized in comprising a VL region of SEQ ID NO:12 and a VH regionselected from the group comprising the VH regions of SEQ ID NO:38, 39,and 40. In one embodiment of the invention the first binding part ischaracterized in comprising a VL region SEQ ID NO:12, wherein amino acid49 is selected from the group of amino acids tyrosine (Y), glutamic acid(E), serine (S), and histidine (H). In one embodiment amino acid 49 isE.

In one embodiment of the invention the first binding part ischaracterized in comprising as VH region a VH region of SEQ ID NO:10. Inone embodiment of the invention the first binding part is characterizedin comprising as VL region a VL region selected from the groupconsisting of VL regions of SEQ ID NO:12, 13, and 14. In one embodimentof the invention the first binding part is characterized in thatcomprising as VH region a VH region of SEQ ID NO:10 and as VL region aVL region of SEQ ID NO:12. In one embodiment of the invention the firstbinding part is characterized in that comprising as VH region a VHregion of SEQ ID NO:10 and as VL region a VL region of SEQ ID NO:13. Inone embodiment of the invention the first binding part is characterizedin that comprising as VH region a VH region of SEQ ID NO:10 and as VLregion a VL region of SEQ ID NO:14.

In one embodiment of the invention the first binding part ischaracterized in comprising as VH region a VH region selected from thegroup consisting of SEQ ID NO:38, 39, and 40. In one embodiment of theinvention the first binding part is characterized in that comprising asVH region a VH region of SEQ ID NO:38 and as VL region a VL region ofSEQ ID NO:12. In one embodiment of the invention the first binding partis characterized in that comprising as VH region a VH region of SEQ IDNO:39 and as VL region a VL region of SEQ ID NO:12. In one embodiment ofthe invention the first binding part is characterized in that comprisingas VH region a VH region of SEQ ID NO:40 and as VL region a VL region ofSEQ ID NO:12.

In one embodiment of the invention the first binding part is furthercharacterized in that it binds also specifically to cynomolgus BCMA. Inone embodiment an antibody of the invention shows regarding binding toBCMA a cyno/human affinity gap between 1.5 and 5 or 1.5 and 10 or 1.5and 16 (table 5).

In one embodiment of the invention the bispecific antibody ischaracterized in that it binds also specifically to cynomolgus CD3. Inone embodiment the bispecific anti-BCMA/anti-CD3 antibody shows acyno/human gap of Mab CD3 between 1.25 and 5 or between 0.8 and 1.0.

In one embodiment of the invention the first binding part is an antibodywith an Fc part or without an Fc part including a multispecificantibody, bispecific antibody, a single chain variable fragment (scFv)such as a bispecific T cells engager, diabody, or tandem scFv, anantibody mimetic such as DARPin, a naked monospecific antibody, or anantibody drug conjugate. In one embodiment a multispecific antibody,bispecific antibody, a bispecific T cells engager, diabody, or tandemscFv is specifically binding to BCMA and CD3.

Based on the first binding part it is possible to generate antibody-drugconjugates against BCMA and multispecific or bispecific antibodiesagainst BCMA and CD3 in different formats with or without an Fc portionknown in the state of the art (see e. g. above in “background of theinvention”), single chain variable fragments (scFv) such as bispecific Tcells engagers, diabodies, tandem scFvs, and antibody mimetics such asDARPins, all of them are also embodiments of the invention. Bispecificantibody formats are well known in the state of the art and e.g. alsodescribed in Kontermann R E, mAbs 4:2 1-16 (2012); Holliger P., Hudson PJ, Nature Biotech. 23 (2005) 1126-1136 and Chan A C, Carter P J NatureReviews Immunology 10, 301-316 (2010) and Cuesta A M et al., TrendsBiotech 28 (2011) 355-362.

In one embodiment of the invention the first binding part ischaracterized in comprising as BCMA VH a VH region of SEQ ID NO:10.

In one embodiment of the invention the first binding part ischaracterized in that the BCMA VL is selected from the group consistingof VL regions of SEQ ID NO:12, 13, and 14. In one embodiment of theinvention the first binding part is characterized in comprising as BCMAVH region a VH region of SEQ ID NO:10 and as VL region a VL region ofSEQ ID NO:12. In one embodiment of the invention the first binding partis characterized in comprising as BCMA VH a VH region of SEQ ID NO:10and as VL region a VL region of SEQ ID NO:13. In one embodiment of theinvention the first binding part is characterized in comprising as BCMAVH a VH region of SEQ ID NO:10 and as VL region a VL region of SEQ IDNO:14.

In one embodiment of the invention the first binding part ischaracterized in comprising a VL region selected from the groupconsisting of VL regions of SEQ ID NO:12, 13, and 14 wherein amino acid49 is selected from the group of amino acids tyrosine (Y), glutamic acid(E), serine (S), and histidine (H). In one embodiment amino acid 49 is E(SEQ ID NO:12), S (SEQ ID NO:13) or H (SEQ ID NO:14). In one embodimentof the invention the first binding part is characterized in comprising aVL region selected from the group consisting of VL regions of SEQ IDNO:12, 13, and 14 wherein amino acid 74 is threonine (T) or alanine (A).In one embodiment amino acid 74 is A within SEQ ID NO:14.

In one embodiment of the invention the first binding part ischaracterized in comprising a BCMA VH comprising a CDR3H region of SEQID NO:17 and a BCMA VL comprising a CDR1L region of SEQ ID NO:31, aCDR2L region of SEQ ID NO:32 and a CDR3L region of SEQ ID NO:20 and aCDR1L and CDR2L region combination selected from the group of

a) CDR1H region of SEQ ID NO:29 and CDR2H region of SEQ ID NO:30,

b) CDR1H region of SEQ ID NO:34 and CDR2H region of SEQ ID NO:35, or

c) CDR1H region of SEQ ID NO:36 and CDR2H region of SEQ ID NO:37.

The bispecific antibody against BCMA and CD3 is characterized in oneembodiment in comprising

-   -   a) a light chain and heavy chain of an antibody specifically        binding to one of said targets CD3 and BCMA; and    -   b) a light chain and heavy chain of an antibody specifically        binding to the other one of said targets, wherein the variable        domains VL and VH or the constant domains CL and CH1 are        replaced by each other.

In one embodiment a VH domain of said anti-CD3 antibody portion islinked to a CH1 or CL domain of said anti-BCMA antibody portion. In oneembodiment a VL domain of said anti-CD3 antibody portion is linked to aCH1 or CL domain of said anti-BCMA antibody portion.

In one embodiment the bispecific antibody comprises not more than oneFab fragment of an anti-CD3 antibody portion, not more than two Fabfragments of an anti-BCMA antibody portion and not more than one Fcpart, in one embodiment a human Fc part. In one embodiment not more thanone Fab fragment of the anti-CD3 antibody portion and not more than oneFab fragment of the anti-BCMA antibody portion are linked to the Fc partand linking is performed via C-terminal binding of the Fab fragment(s)to the hinge region. In one embodiment the second Fab fragment of theanti-BCMA antibody portion is linked via its C-terminus either to theN-terminus of the Fab fragment of the anti-CD3 antibody portion or tothe hinge region of the Fc part and therefore between the Fc part andthe anti-CD3 antibody portion. The preferred bispecific antibodies areshown in FIGS. 1 to 3.

Especially preferred are bispecific antibodies comprising only the Fabfragments and the Fc part as specified, with or without “aasubstitution”:

Fab BCMA-Fc-Fab CD3 (bispecific format FIG. 1A or 1B),

Fab BCMA-Fc-Fab CD3-Fab BCMA (bispecific format FIG. 2A or 2B),

Fab BCMA-Fc-Fab BCMA-Fab CD3 (bispecific format FIG. 2C or 2D),

Fc-Fab CD3-Fab BCMA (bispecific format FIG. 3A or 3B),

Fc-Fab BCMA-Fab CD3 (bispecific format FIG. 3C or 3D).

As shown in FIGS. 1 to 3 “Fab BCMA-Fc, “Fab BCMA-Fc-Fab CD3” and “FabBCMA-Fc-Fab CD3” means that the Fab fragment(s) is (are) bound via its(their) C-terminus to the N-terminus of the Fc fragment. “Fab CD3-FabBCMA” means that the Fab CD3 fragment is bound with its N-terminus tothe C-terminus of the Fab BCMA fragment. “Fab BCMA—Fab CD3” means thatthe Fab BCMA fragment is bound with its N-terminus to the C-terminus ofthe Fab CD3 fragment.

In one embodiment the bispecific antibody comprises a second Fabfragment of said anti-BCMA antibody linked with its C-terminus to theN-terminus of the CD3 antibody portion of said bispecific antibody. Inone embodiment a VL domain of said first anti-CD3 antibody portion islinked to a CH1 or CL domain of said second anti-BCMA antibody.

In one embodiment the bispecific antibody comprises a second Fabfragment of said anti-BCMA antibody linked with its C-terminus to the Fcpart (like the first Fab fragment of said anti-BCMA antibody) and linkedwith its N-terminus to the C-terminus of the CD3 antibody portion. Inone embodiment a CH1 domain of said anti-CD3 antibody portion is linkedto the VH domain of said second anti-BCMA antibody portion.

In one embodiment the bispecific antibody comprises an Fc part linkedwith its N-terminus to the C-terminus of said CD3 antibody Fab fragment.In one embodiment the bispecific antibody comprises an Fc part linkedwith its first N-terminus to the C-terminus of said CD3 antibody Fabfragment and a second Fab fragment of said anti-BCMA antibody linkedwith its C-terminus to the second N-terminus of the Fc part.

In one embodiment the CL domain of the CD3 antibody Fab fragment islinked to the hinge region of the Fc part. In one embodiment the CH1domain of the BCMA antibody Fab fragment is linked to the hinge regionof the Fc part.

The Fab fragments are linked together by the use of an appropriatelinker according to the state of the art. In one embodiment a(Gly4-Ser1)3 linker is used (Desplancq D K et al., Protein Eng. 1994August; 7(8):1027-33 and Mack M. et al., PNAS Jul. 18, 1995 vol. 92 no.15 7021-7025). As the linker is a peptidic linker, such covalent bindingis usually performed by biochemical recombinant means, using a nucleicacid encoding the VL and/or VH domains of the respective Fab fragments,the linker and if appropriate the Fc part chain.

In one embodiment of the invention the bispecific antibody against BCMAand CD3 is characterized in that the variable domain VH of the anti-CD3antibody portion (further named as “CD3 VH”) comprises the heavy chainCDRs of SEQ ID NO: 1, 2 and 3 as respectively heavy chain CDR1H, CDR2Hand CDR3H and the variable domain VL of the anti-CD3 antibody portion(further named as “CD3 VL”) comprises the light chain CDRs of SEQ ID NO:4, 5 and 6 as respectively light chain CDR1L, CDR2L and CDR3L.

In one embodiment of the invention the bispecific antibody ischaracterized in that the variable domains of the anti CD3ε antibodyportion are of SEQ ID NO:7 and 8.

In one embodiment of the invention the bispecific antibody ischaracterized in that the anti-CD3 antibody portion (the second bindingpart of the bispecific antibody) is linked at its N-terminus to theC-terminus of a of the anti-BCMA antibody portion (the first bindingpart of the bispecific antibody) and the variable domains VL and VH ofthe anti-CD3 antibody portion or the constant domains CL and CH1 arereplaced by each other.

In one embodiment the VH domain of said anti-CD3 antibody portion islinked to a CH1 or CL domain of said anti-BCMA antibody portion. In oneembodiment a VL domain of said anti-CD3 antibody portion is linked to aCH1 or CL domain of said anti-BCMA antibody portion.

Such antibody portion is in one embodiment a Fab fragment of therespective antibody.

In a further embodiment of the invention the bispecific antibody whereinthe variable domains VL and VH in the light chain and the respectiveheavy chain of the anti-CD3 antibody portion or the anti-BCMA antibodyportion are replaced by each other, is characterized in comprising aconstant domain CL of the anti-CD3 antibody portion or the anti-BCMAantibody portion wherein the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat), and in the respective constant domain CH1 the aminoacid at position 147 and the amino acid at position 213 is substitutedindependently by glutamic acid (E), or aspartic acid (D). In oneembodiment the antibody is monovalent for CD3 binding. In one embodimentin addition to the amino acid replacement at position 124 in theconstant domain CL the amino acid at position 123 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (furthercalled as “charge variant exchange”). In one embodiment the antibody ismonovalent for CD3 binding and amino acid 124 is K, amino acid 147 is E,amino acid 213 is E, and amino acid 123 is R. In one embodiment thebispecific antibody comprises in addition the same anti-BCMA bindingportion once more (in one embodiment a Fab fragment). That means also,that if the first anti-BCMA binding portion comprises the charge variantexchange, then the second anti-BCMA binding portion comprise the samecharge variant exchange. (All amino acid numbering is according toKabat).

In one embodiment of the invention the bispecific antibody ischaracterized in comprising

a) the first light chain and the first heavy chain of a first antibodywhich specifically binds to BCMA; and

b) the second light chain and the second heavy chain of a secondantibody which specifically binds to CD3, and wherein the variabledomains VL and VH in the second light chain and second heavy chain ofthe second antibody are replaced by each other; and

c) wherein in the constant domain CL of the first light chain under a)the amino acid at position 124 is substituted independently by lysine(K), arginine (R) or histidine (H) (numbering according to Kabat), andwherein in the constant domain CH1 of the first heavy chain under a) theamino acid at position 147 and the amino acid at position 213 issubstituted independently by glutamic acid (E), or aspartic acid (D)(numbering according to Kabat) (see e.g. FIGS. 1A, 2A, 2C, 3A, 3C).

In one embodiment said bispecific antibody described in the lastpreceding paragraph is further characterized in that said bispecificantibody comprises in addition a Fab fragment of said first antibody(further named also as “BCMA-Fab”) and in the constant domain CL saidBCMA-Fab the amino acid at position 124 is substituted independently bylysine (K), arginine (R) or histidine (H) (numbering according toKabat), and wherein in the constant domain CH1 of said BCMA-Fab theamino acid at positions 147 and the amino acid at position 213 issubstituted independently by glutamic acid (E), or aspartic acid (D)(numbering according to Kabat) (see e.g. FIGS. 2A, 2C).

In one embodiment of the invention the bispecific antibody ischaracterized in comprising

a) the first light chain and the first heavy chain of a first antibodywhich specifically binds to BCMA; and

b) the second light chain and the second heavy chain of a secondantibody which specifically binds to CD3, and wherein the variabledomains VL and VH in the second light chain and second heavy chain ofthe second antibody are replaced by each other; and wherein

c) in the constant domain CL of the second light chain under b) theamino acid at position 124 is substituted independently by lysine (K),arginine (R) or histidine (H) (numbering according to Kabat), andwherein in the constant domain CH1 of the second heavy chain under b)the amino acid at positions 147 and the amino acid at position 213 issubstituted independently by glutamic acid (E), or aspartic acid (D)(numbering according to Kabat).

In one embodiment in addition to the amino acid replacement at position124 in the constant domain CL of the first or second light chain theamino acid at position 123 is substituted independently by lysine (K),arginine (R) or histidine (H).

In one embodiment in the constant domain CL the amino acid at position124 is substituted by lysine (K), in the constant domain CH1 the aminoacid at position 147 and the amino acid at position 213 are substitutedby glutamic acid (E). In one embodiment in addition in the constantdomain CL in the amino acid at position 123 is substituted by arginine(R).

In one embodiment of the invention the bispecific antibody consists ofone Fab fragment of an antibody specifically binding to CD3 (furthernamed also as “CD3-Fab”), and one Fab fragment of an anti-BCMA antibody(further named also as “BCMA-Fab(s)”) and a Fc part, wherein the CD3-Faband the BCMA-Fab are linked via their C-termini to the hinge region ofsaid Fc part. Either the CD3-Fab or the BCMA-Fab comprises aasubstitution and the CD3-Fab comprises crossover (FIGS. 1A and 1B).

In one embodiment of the invention the bispecific antibody consists ofone CD3-Fab, and one BCMA-Fab and a Fc part, wherein the CD3-Fab and theBCMA-Fab are linked via their C-termini to the hinge region of said Fcpart and a second BCMA-Fab, which is linked with its C-terminus to theN-terminus of the CD3-Fab. The CD3-Fab comprises crossover and eitherthe CD3-Fab or both BCMA-Fabs comprise aa substitution (FIGS. 2A and2B). Especially preferred is a bispecific antibody comprisingBCMA-Fab-Fc-CD3-Fab-BCMA-Fab, wherein both BCMA-Fabs comprise aasubstitution and the CD3-Fab comprises VL/VH crossover (FIG. 2A).Especially preferred is a bispecific antibody consisting ofBCMA-Fab-Fc-CD3-Fab-BCMA-Fab, wherein both BCMA-Fabs comprise aasubstitution Q124K, E123R, K147E and K213E and the CD3-Fab comprisesVL/VH crossover. Especially preferred is that both BCMA-Fabs comprise asCDRs the CDRs of antibody 21, 22, or 42, or as VH/VL the VH/VL ofantibody 21, 22, or 42 (for antibodies 21, 22 and 42 see table 1A and Blater in the text)

In one embodiment of the invention the bispecific antibody consists oftwo BCMA-Fabs and an Fc part, wherein one BCMA-Fab and the CD3 Fab arelinked via their C-termini to the hinge region of said Fc part and thesecond BCMA-Fab is linked with its C-terminus to the N-terminus of theCD3-Fab. The CD3-Fab comprises crossover and either the CD3-Fab or bothBCMA-Fabs comprise aa substitution (FIGS. 2A and 2B).

In one embodiment of the invention the bispecific antibody consists oftwo BCMA-Fabs and an Fc part, wherein the BCMA-Fabs are linked via theirC-termini to the hinge region of said Fc part and a CD3-Fab, which islinked with its C-terminus to the N-terminus of one BCMA-Fab. TheCD3-Fab comprises crossover and either the CD3-Fab or both BCMA-Fabscomprise aa substitution (FIGS. 2C and 2D).

In one embodiment of the invention the bispecific antibody consists ofone CD3-Fab, which is linked via its C-terminus to the hinge region ofsaid Fc part and a BCMA-Fab, which is linked with its C-terminus to theN-terminus of the CD3-Fab. The CD3-Fab comprises crossover and eitherthe CD3-Fab or the BCMA-Fab comprise aa substitution (FIGS. 1A and 1B).

In one embodiment of the invention the bispecific antibody consists ofone CD3-Fab, which is linked via its C-terminus to the hinge region ofsaid Fc part and a BCMA-Fab, which is linked with its C-terminus to theN-terminus of the CD3-Fab. The CD3-Fab comprises crossover and eitherthe CD3-Fab or the BCMA-Fab comprise aa substitution (FIGS. 3A and 3B).

In one embodiment of the invention the bispecific antibody consists ofone BCMA-Fab, which is linked via its C-terminus to the hinge region ofsaid Fc part and a CD3-Fab, which is linked with its C-terminus to theN-terminus of the BCMA-Fab. The CD3-Fab comprises crossover and eitherthe CD3-Fab or the BCMA-Fab comprise aa substitution (FIGS. 3C and 3D).

The Fab fragments are chemically linked together by the use of anappropriate linker according to the state of the art. In one embodimenta (Gly4-Ser1)3 linker is used (Desplancq D K et al., Protein Eng. 1994August; 7(8):1027-33 and Mack M. et al., PNAS Jul. 18, 1995 vol. 92 no.15 7021-7025). Linkage between two Fab fragments is performed betweenthe heavy chains. Therefore the C-terminus of CH1 of a first Fabfragment is linked to the N-terminus of VH of the second Fab fragment(no crossover) or to VL (crossover). Linkage between a Fab fragment andthe Fc part is performed as linkage between CH1 and CH2.

The first and a second Fab fragment of an antibody specifically bindingto BCMA are in one embodiment derived from the same antibody and in oneembodiment identical in the CDR sequences, variable domain sequences VHand VL and/or the constant domain sequences CH1 and CL. In oneembodiment the amino acid sequences of the first and a second Fabfragment of an antibody specifically binding to BCMA are identical. Inone embodiment the BCMA antibody is an antibody comprising the CDRsequences of antibody 21, 22, or 42, an antibody comprising the VH andVL sequences of antibody 21, 22, or 42, or an antibody comprising theVH, VL, CH1, and CL sequences of antibody 21, 22, or 42.

In one embodiment the bispecific antibody comprises as Fab fragments andFc part, not more than one Fab fragment of an anti-CD3 antibody, notmore than two Fab fragments of an anti-BCMA antibody and not more thanone Fc part, in one embodiment a human Fc part. In one embodiment thesecond Fab fragment of an anti-BCMA antibody is linked via itsC-terminus either to the N-terminus of the Fab fragment of an anti-CD3antibody or to the hinge region of the Fc part. In one embodimentlinkage is performed between CH1 of BCMA-Fab and VL of CD3-Fab (VL/VHcrossover).

In one embodiment the antibody portion specifically binding to humanCD3, in one embodiment the Fab fragment, is characterized in comprisinga variable domain VH comprising the heavy chain CDRs of SEQ ID NO: 1, 2and 3 as respectively heavy chain CDR1, CDR2 and CDR3 and a variabledomain VL comprising the light chain CDRs of SEQ ID NO: 4, 5 and 6 asrespectively light chain CDR1, CDR2 and CDR3 of the anti-CD3ε antibody(CDR MAB CD3). In one embodiment the antibody portion specificallybinding to human CD3 is characterized in that the variable domains areof SEQ ID NO:7 and 8 (VHVL MAB CD3).

In one embodiment of the invention the bispecific antibody isspecifically binding to the extracellular domain of human BCMA and tohuman CD3ε, characterized in comprising a heavy and light chain setselected from the group consisting of polypeptides

i) SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, and SEQ ID NO:51 (2×); (set1 TCB of antibody 21),

ii) SEQ ID NO:48, SEQ ID NO:52, SEQ ID NO:53, and SEQ ID NO:54 (2×) (set2 TCB of antibody 22), and

iii) SEQ ID NO:48, SEQ ID NO:55, SEQ ID NO:56, and SEQ ID NO:57 (2×)(set 3 TCB of antibody 42).

In one embodiment of the invention the bispecific antibody ischaracterized in that the CH3 domain of one heavy chain and the CH3domain of the other heavy chain each meet at an interface whichcomprises an original interface between the antibody CH3 domains;wherein said interface is altered to promote the formation of thebispecific antibody, wherein the alteration is characterized in that:

-   -   a) the CH3 domain of one heavy chain is altered, so that within        the original interface the CH3 domain of one heavy chain that        meets the original interface of the CH3 domain of the other        heavy chain within the bispecific antibody, an amino acid        residue is replaced with an amino acid residue having a larger        side chain volume, thereby generating a protuberance within the        interface of the CH3 domain of one heavy chain which is        positionable in a cavity within the interface of the CH3 domain        of the other heavy chain and    -   b) the CH3 domain of the other heavy chain is altered, so that        within the original interface of the second CH3 domain that        meets the original interface of the first CH3 domain within the        bispecific antibody an amino acid residue is replaced with an        amino acid residue having a smaller side chain volume, thereby        generating a cavity within the interface of the second CH3        domain within which a protuberance within the interface of the        first CH3 domain is positionable.

In one embodiment of the invention the bispecific antibody ischaracterized in that said amino acid residue having a larger side chainvolume is selected from the group consisting of arginine (R),phenylalanine (F), tyrosine (Y), tryptophan (W).

In one embodiment of the invention the bispecific antibody ischaracterized in that said amino acid residue having a smaller sidechain volume is selected from the group consisting of alanine (A),serine (S), threonine (T), valine (V).

In one embodiment such a bispecific antibody is characterized in thatboth CH3 domains are further altered by the introduction of cysteine (C)as amino acid in the corresponding positions of each CH3 domain.

In one embodiment such a bispecific antibody is characterized in thatone of the constant heavy chain domains CH3 of both heavy chains isreplaced by a constant heavy chain domain CH1; and the other constantheavy chain domain CH3 is replaced by a constant light chain domain CL.

In one embodiment of the invention the bispecific antibody comprises amodified Fc part inducing cell death of 20% or more cells of apreparation of BCMA expressing cells after 24 hours at a concentrationof said antibody of 100 nM by ADCC relative to a control under identicalconditions using the same antibody with the parent Fc part as control.Such an antibody is in one embodiment a naked antibody.

In one embodiment of the invention the bispecific antibody is anantibody with an amount of fucose of 60% or less of the total amount ofoligosaccharides (sugars) at Asn297 (see e.g. US20120315268).

In one embodiment the Fc part comprises the amino acid substitutionswhich are introduced in a human Fc part and disclosed in SEQ ID NO:55and 56.

A further embodiment of the invention is a chimeric antigen receptor(CAR) or the respective CAR T-cell of an anti-BCMA antibody togetherwith an immunotherapeutic drug selected from the group consisting ofthalidomide and an immunotherapeutic derivative thereof, an anti-CD38antibody, an anti-PD-1 antibody and an anti-PD-L1 antibody, for combineduse in treating multiple myeloma. In such an embodiment the anti-BCMAantibody consists of a single chain VH and VL domain of the firstbinding part and a CD3-zeta transmembrane and endodomain Preferably theCD3 zeta domain is linked via a spacer with the C-terminus of said VLdomain and the N terminus of the VL domain is linked via a spacer to theC terminus of said VH domain. Chimeric antigen receptors of BCMAantibodies, useful transmembrane domains and endodomains, and methodsfor the production are described e.g. in Ramadoss NS. et al., J. Am.Chem. Soc. J., DOI: 10.1021/jacs.5b01876 (2015), Carpenter R O et al.,Clin. Cancer. Res. DOI: 10.1158/1078-0432.CCR-12-2422 (2013),WO2015052538 and WO2013154760.

In one embodiment of the invention the first binding part is Mab21,Mab22, Mab42, Mab27, Mab33, and Mab39 (for antibodies Mab 21, 22, 42,27, 33, 39 see table 1A and B later in the text) as described herein bytheir CDR sequences, and/or VH/VL sequences together with the describedCL and CH1 sequences, as antigen binding fragments, especially Fabfragments. In one embodiment the bispecific antibody comprises an Fcpart or not, especially the 2+1 format, and the heavy and light chainsof the bispecific antibody is especially as described in table 1A.

The anti-BCMA antibody depletes, in the bispecific format, especially inthe 2+1 format, human malignant plasma cells in Multiple Myeloma MM bonemarrow aspirates to at least 80% after a 48 hour treatment in aconcentration of between 10 nM and 1 fM inclusively. The anti-BCMAantibodies have been characterized in panning a variable heavy chain(VH) and a variable light chain (VL) phage-display library of antibody83A10 (VH library, VL library) with 1-50 nM cyno BCMA in 1-3 rounds andselecting a variable light chain and a variable heavy chain which havesuch properties as such bispecific T cell binder. Preferably panning isperformed in 3 rounds, using 50 nM cynoBCMA for round 1, 25 nM cyBCMAfor round 2 and 10 nM cyBCMA for round 3. Preferably the libraries arerandomized in either the light chain CDR1 and CDR2 or the heavy chainCDR1 and CDR2. Preferably a light and heavy chain are identified whicheach bind as Fab fragment, comprising in addition the corresponding VHor VL of antibody 83A10, to huBCMA with a Kd of 50 pM to 5 nM and tocyno BCMA with a Kd of 0.1 nM to 20 nM. Preferably the bispecific formatis the format of FIG. 2A, comprising the respective constant domains VLand VH of the CD3 Fab replacement by each other and within both BCMAFabs amino acid exchanges K213E and K147E in the CH1 domain and aminoacid exchanges E123R and Q124K in the CL domain

The bispecific antibody as mentioned herein can be prepared by the stepsof

-   -   a) transforming a host cell with    -   b) vectors comprising nucleic acid molecules encoding the light        chain and heavy chain of said antibody molecule,    -   c) culturing the host cell under conditions that allow synthesis        of said antibody molecule; and    -   d) recovering said antibody molecule from said culture.

The bispecific antibody as mentioned herein can be prepared by the stepsof

-   -   e) transforming a host cell with    -   f) vectors comprising nucleic acid molecules encoding the light        chain and heavy chain of an antibody specifically binding to the        first target    -   g) vectors comprising nucleic acid molecules encoding the light        chain and heavy chain of an antibody specifically binding to the        second target, wherein the variable domains VL and VH or the        constant domains CL and CH1 are replaced by each other;    -   h) culturing the host cell under conditions that allow synthesis        of said antibody molecule; and    -   i) recovering said antibody molecule from said culture.

A further embodiment of the invention is a pharmaceutical compositioncomprising a bispecific antibody specifically binding to human B cellmaturation antigen (BCMA) and to human CD3ε (CD3) together with animmunotherapeutic drug selected from the group consisting of thalidomideand an immunotherapeutic derivative thereof, an anti-CD38 antibody, ananti-PD-1 antibody and an anti-PD-L1 antibody, for combined use intreating multiple myeloma and a pharmaceutically acceptable excipient.

A further embodiment of the invention is a pharmaceutical compositioncomprising a bispecific antibody specifically binding to human B cellmaturation antigen (BCMA) and to human CD3ε (CD3) together with animmunotherapeutic drug selected from the group consisting of thalidomideand an immunotherapeutic derivative thereof, an anti-CD38 antibody, ananti-PD-1 antibody and an anti-PD-L1 antibody, for combined use intreating multiple myeloma for use as a medicament.

A further embodiment of the invention is a pharmaceutical composition,comprising a bispecific antibody specifically binding to human B cellmaturation antigen (BCMA) and to human CD3ε (CD3) together with animmunotherapeutic drug selected from the group consisting of thalidomideand an immunotherapeutic derivative thereof, an anti-CD38 antibody, ananti-PD-1 antibody and an anti-PD-L1 antibody, for combined use intreating multiple myeloma, characterized in that said first binding partcomprises a VH region comprising a CDR1H region of SEQ ID NO:21, a CDR2Hregion of SEQ ID NO:22 and a CDR3H region of SEQ ID NO:17 and a VLregion comprising a CDR3L region of SEQ ID NO:20 and a CDR1L and CDR2Lregion combination selected from the group of

i) CDR1L region of SEQ ID NO:23 and CDR2L region of SEQ ID NO:24,

ii) CDR1L region of SEQ ID NO:25 and CDR2L region of SEQ ID NO:26, or

iii) CDR1L region of SEQ ID NO:27 and CDR2L region of SEQ ID NO:28. Afurther embodiment of the invention is a pharmaceutical composition,comprising a first binding part specifically binding to human B cellmaturation antigen (BCMA) and a second binding part specifically bindingto human CD3ε (CD3), together with an immunotherapeutic drug selectedfrom the group consisting of thalidomide and an immunotherapeuticderivative thereof, an anti-CD38 antibody, an anti-PD-1 antibody and ananti-PD-L1 antibody, for combined use as a medicament in the treatmentof Multiple Myeloma.

In one embodiment a bispecific antibody according to the invention,comprising a first binding part specifically binding to human B cellmaturation antigen (BCMA) and a second binding part specifically bindingto human CD3ε (CD3), together with an immunotherapeutic drug selectedfrom the group consisting of thalidomide and an immunotherapeuticderivative thereof, an anti-CD38 antibody, an anti-PD-1 antibody and ananti-PD-L1 antibody, for combined use can be used for the treatment ofplasma cell disorders like Multiple Myeloma MM or other plasma celldisorders expressing BCMA as described below. MM is a plasma cellmalignancy characterized by a monoclonal expansion and accumulation ofabnormal plasma cells in the bone marrow compartment. MM also involvescirculating clonal plasma cells with same IgG gene rearrangement andsomatic hypermutation. MM arises from an asymptomatic, premalignantcondition called monoclonal gammopathy of unknown significance (MGUS),characterized by low levels of bone marrow plasma cells and a monoclonalprotein. MM cells proliferate at low rate. MM results from a progressiveoccurrence of multiple structural chromosomal changes (e.g. unbalancedtranslocations). MM involves the mutual interaction of malignant plasmacells and bone marrow microenvironment (e.g. normal bone marrow stromalcells). Clinical signs of active MM include monoclonal antibody spike,plasma cells overcrowding the bone marrow, lytic bone lesions and bonedestruction resulting from overstimulation of osteoclasts (Dimopulos &Terpos, Ann Oncol 2010; 21 suppl 7: vii 143-150). Another plasma celldisorder involving plasma cells i.e. expressing BCMA is systemic lupuserythematosus (SLE), also known as lupus. SLE is a systemic, autoimmunedisease that can affect any part of the body and is represented with theimmune system attacking the body's own cells and tissue, resulting inchronic inflammation and tissue damage. It is a Type IIIhypersensitivity reaction in which antibody-immune complexes precipitateand cause a further immune response (Inaki & Lee, Nat Rev Rheumatol2010; 6: 326-337). Further plasma cell disorders are plasma cellleukemia and AL-Amyloidosis (see also Examples 19 and 20). In all theseplasma cell disorders depletion of plasma cells/malignant plasma cellsby antibodies according to this invention is expected to be beneficialfor the patients suffering from such a disease.

A further embodiment of the invention is a bispecific antibodycomprising a first binding part specifically binding to human B cellmaturation antigen (BCMA) and a second binding part specifically bindingto human CD3ε (CD3), together with an immunotherapeutic drug selectedfrom the group consisting of thalidomide and an immunotherapeuticderivative thereof, an anti-CD38 antibody, an anti-PD-1 antibody and ananti-PD-L1 antibody, for combined use as a medicament. Preferably thefirst binding part is characterized in that said first binding partcomprises a VH region comprising a CDR1H region of SEQ ID NO:21, a CDR2Hregion of SEQ ID NO:22 and a CDR3H region of SEQ ID NO:17 and a VLregion comprising a CDR3L region of SEQ ID NO:20 and a CDR1L and CDR2Lregion combination selected from the group of

i) CDR1L region of SEQ ID NO:23 and CDR2L region of SEQ ID NO:24,

ii) CDR1L region of SEQ ID NO:25 and CDR2L region of SEQ ID NO:26, or

iii) CDR1L region of SEQ ID NO:27 and CDR2L region of SEQ ID NO:28.

A further embodiment of the invention is a pharmaceutical compositioncomprising a bispecific antibody comprising a first binding partspecifically binding to human B cell maturation antigen (BCMA) and asecond binding part specifically binding to human CD3ε (CD3), togetherwith an immunotherapeutic drug selected from the group consisting ofthalidomide and an immunotherapeutic derivative thereof, an anti-CD38antibody, an anti-PD-1 antibody and an anti-PD-L1 antibody, for use as amedicament. Preferably the first binding part is characterized in thatsaid first binding part comprises a VH region comprising a CDR1H regionof SEQ ID NO:21, a CDR2H region of SEQ ID NO:22 and a CDR3H region ofSEQ ID NO:17 and a VL region comprising a CDR3L region of SEQ ID NO:20and a CDR1L and CDR2L region combination selected from the group of

i) CDR1L region of SEQ ID NO:23 and CDR2L region of SEQ ID NO:24,

ii) CDR1L region of SEQ ID NO:25 and CDR2L region of SEQ ID NO:26, or

iii) CDR1L region of SEQ ID NO:27 and CDR2L region of SEQ ID NO:28.

A further embodiment of the invention is a pharmaceutical compositioncomprising said bispecific antibody with increased effector functiontogether with an immunotherapeutic drug selected from the groupconsisting of thalidomide and an immunotherapeutic derivative thereof,an anti-CD38 antibody, an anti-PD-1 antibody and an anti-PD-L1 antibody,for use as a medicament. Preferably the first binding part ischaracterized in that said first binding part comprises a VH regioncomprising a CDR1H region of SEQ ID NO:21, a CDR2H region of SEQ IDNO:22 and a CDR3H region of SEQ ID NO:17 and a VL region comprising aCDR3L region of SEQ ID NO:20 and a CDR1L and CDR2L region combinationselected from the group of

i) CDR1L region of SEQ ID NO:23 and CDR2L region of SEQ ID NO:24,

ii) CDR1L region of SEQ ID NO:25 and CDR2L region of SEQ ID NO:26, or

iii) CDR1L region of SEQ ID NO:27 and CDR2L region of SEQ ID NO:28.

A further embodiment of the invention is a pharmaceutical compositioncomprising said bispecific antibody with decreased effector functiontogether with an immunotherapeutic drug selected from the groupconsisting of thalidomide and an immunotherapeutic derivative thereof,an anti-CD38 antibody, an anti-PD-1 antibody and an anti-PD-L1 antibodyfor use as a medicament. Preferably the first binding part ischaracterized in that said first binding part comprises a VH regioncomprising a CDR1H region of SEQ ID NO:21, a CDR2H region of SEQ IDNO:22 and a CDR3H region of SEQ ID NO:17 and a VL region comprising aCDR3L region of SEQ ID NO:20 and a CDR1L and CDR2L region combinationselected from the group of

i) CDR1L region of SEQ ID NO:23 and CDR2L region of SEQ ID NO:24,

ii) CDR1L region of SEQ ID NO:25 and CDR2L region of SEQ ID NO:26, or

iii) CDR1L region of SEQ ID NO:27 and CDR2L region of SEQ ID NO:28.

A further embodiment of the invention is a pharmaceutical compositioncomprising said bispecific antibody as a diabody together with animmunotherapeutic drug selected from the group consisting of thalidomideand an immunotherapeutic derivative thereof, an anti-CD38 antibody, ananti-PD-1 antibody and an anti-PD-L1 antibody for use as a medicament.Preferably the first binding part is characterized in that said firstbinding part comprises a VH region comprising a CDR1H region of SEQ IDNO:21, a CDR2H region of SEQ ID NO:22 and a CDR3H region of SEQ ID NO:17and a VL region comprising a CDR3L region of SEQ ID NO:20 and a CDR1Land CDR2L region combination selected from the group of

i) CDR1L region of SEQ ID NO:23 and CDR2L region of SEQ ID NO:24,

ii) CDR1L region of SEQ ID NO:25 and CDR2L region of SEQ ID NO:26, or

iii) CDR1L region of SEQ ID NO:27 and CDR2L region of SEQ ID NO:28.

In one embodiment the bispecific antibody and the immunotherapeutic drugselected from the group consisting of thalidomide and animmunotherapeutic derivative thereof, an anti-CD38 antibody, ananti-PD-1 antibody and an anti-PD-L1 antibody, are administered once ortwice a week in one embodiment via subcutaneous administration (e.g. inone embodiment in the dose range of 0.1 to 2.5, preferably to 25mg/m²/week, preferably to 250 mg/m²/week). Due to superior cytotoxicityactivities of the bispecific antibody they can be administered at leastat the same magnitude of clinical dose range (or even lower) as comparedto conventional monospecific antibodies or conventional bispecificantibodies that are not T cell bispecifics (i.e. do not bind to CD3 onone arm). It is envisaged that for a bispecific antibody and theimmunotherapeutic drug selected from the group consisting of thalidomideand an immunotherapeutic derivative thereof, an anti-CD38 antibody, ananti-PD-1 antibody and an anti-PD-L1 antibody subcutaneousadministration is preferred in the clinical settings (e.g. in the doserange of 0.1-250 mg/m²/week). In addition, in patients with high levelsof serum APRIL and BAFF (e.g. multiple myeloma patients) it may not berequired to increase the dose for the bispecific antibody as it may notbe affected by ligand competition. In contrast, the doses for otherligand-blocking/competing anti-BCMA antibodies may need to be increasedin those patients. Another advantage of the bispecific antibody is anelimination half-life of about 4 to 12 days which allows at least onceor twice/week administration.

In one embodiment the bispecific antibody is an antibody with propertiesallowing for once/twice a week treatment by intravenous route butpreferably via subcutaneous administration (e.g. a dosage in the rangeof 200-2000 mg/m/week for 4 weeks). It is envisaged that for thebispecific antibody subcutaneous administration is possible andpreferred in the clinical settings (e.g. in the dose range of 200-2000mg/m²/week depending on the disease indications). In addition, inpatients with high levels of serum APRIL and BAFF (e.g. multiple myelomapatients) it may not be required to increase the dose for the bispecificantibody (e.g. non-ligand blocking/competing antibody) as it may not beaffected by ligand competition. In contrast, the doses for otherligand-blocking/competing anti-BCMA antibodies may need to be increasedin those patients, making subcutaneous administration technically morechallenging (e.g. pharmaceutical). Another advantage of the bispecificantibody is based on the inclusion of an Fc portion, which is associatedwith an elimination half-life of 4 to 12 days and allows at least onceor twice/week administration.

DESCRIPTION OF THE FIGURES

FIGS. 1A-1B. Bispecific bivalent antibodies comprising only the Fabfragments (specific to CD3 and BCMA) and the Fc part as specified: (FIG.1A) Fab BCMA(RK/EE)-Fc-Fab CD3; (FIG. 1B) Fab BCMA-Fc-Fab CD3(RK/EE). aasubstitutions for RK/EE introduced in CL-CH1 to reduce LCmispairing/side products in production. The Fab CD3 includes a VL-VHcrossover to reduce LC mispairing and side-products.

FIGS. 2A-2D. Preferred bispecific trivalent antibodies comprising onlythe Fab fragments (specific to CD3 and BCMA) and the Fc part asspecified: (FIG. 2A) Fab BCMA(RK/EE)-Fc-Fab CD3-Fab BCMA(RK/EE); (FIG.2B) Fab BCMA-Fc-Fab CD3(RK/EE)-Fab BCMA; (FIG. 2C) FabBCMA(RK/EE)-Fc-Fab BCMA(RK/EE)-Fab CD3; (FIG. 2D) Fab BCMA-Fc-FabBCMA-Fab CD3(RK/EE). aa substitutions for RK/EE introduced in CL-CH1 toreduce LC mispairing/side-products in production. Preferably, the FabCD3 includes a VL-VH crossover to reduce LC mispairing andside-products. Preferably, Fab CD3 and Fab BCMA are linked to each otherwith flexible linkers.

FIGS. 3A-3D. Bispecific bivalent antibodies comprising only the Fabfragments (specific to CD3 and BCMA) and the Fc part as specified: (FIG.3A) Fc-Fab CD3-Fab BCMA(RK/EE); (FIG. 3B) Fc-Fab CD3(RK/EE)-Fab BCMA;(FIG. 3C) Fc-Fab BCMA(RK/EE)-Fab CD3; (FIG. 3D) Fc-Fab BCMA-FabCD3(RK/EE). Preferably, the Fabs CD3 include a VL-VH crossover to reduceLC mispairing and side-products. Fab CD3 and Fab BCMA are linked to eachother with flexible linkers.

FIGS. 4A-4D. Redirected T-cell lysis of H929 MM cells induced byanti-BCMA/anti-CD3 T-cell bispecific antibodies (BCMA×CD3-TCB) asmeasured by LDH release. Concentration response curves for lysis of H929MM cells induced by 21-TCBcv (closed circle), 22-TCBcv (closedtriangle), 42-TCBcv (closed square) in comparison with 83A10-TCBcv (opencircle, dotted line). For the definition of the term TCB or TCBcv seeabove. TCBcv used in the experiments for which results are shown inFIGS. 4 to 16 and 19 to 21 had the format shown in FIG. 2A (cv meanscharge variant aa substitutions). There was a concentration-dependentkilling of H929 cells for all anti-BCMA/anti-CD3 T cell bispecificantibodies while no killing was observed with the control-TCB.Experiments were performed with PBMC donor 1 (FIG. 4A), donor 3 (FIG.4B), donor 4 (FIG. 4C), donor 5 (FIG. 4D) using an effector cell totumor target cell (E:T) ratio of 10 PBMCs to 1 MM cell (see example 8).

FIGS. 5A-5E. Redirected T-cell lysis of L363 MM cells induced byanti-BCMA/anti-CD3 T-cell bispecific antibodies as measured by LDHrelease. Concentration response curves for lysis of L363 MM cellsinduced by 21-TCBcv (closed circle), 22-TCBcv (closed triangle),42-TCBcv (closed square) in comparison with 83A10-TCBcv (open circle,dotted line). A concentration-dependent killing of L363 cells wasobserved for all anti-BCMA/anti-CD3 T cell bispecific antibodies whileno killing was observed with the control-TCB. Experiments were performedwith PBMC donor 1 (FIG. 5A), donor 2 (FIG. 5B), donor 3 (FIG. 5C), donor4 (FIG. 5D), donor 5 (FIG. 5E) using an E:T ratio of 10 PBMCs to 1 MMcell (see example 9).

FIGS. 6A-6D. Redirected T-cell lysis of RPMI-8226 MM cells induced byanti-BCMA/anti-CD3 T-cell bispecific antibodies as measured by LDHrelease. Concentration response curves for lysis of RPMI-8226 MM cellsinduced by 21-TCBcv (closed circle), 22-TCBcv (closed triangle),42-TCBcv (closed square) in comparison with 83A10-TCBcv (open circle,dotted line). A concentration-dependent killing of RPMI-8226 cells wasobserved for all anti-BCMA/anti-CD3 T cell bispecific antibodies whileno killing was observed with the control-TCB. Experiments were performedwith PBMC donor 2 (FIG. 6A), donor 3 (FIG. 6B), donor 4 (FIG. 6C), donor5 (FIG. 6D) using an E:T ratio of 10 PBMCs to 1 MM cell (see example10).

FIGS. 7A-7D. Redirected T-cell lysis of JJN-3 MM cells induced byanti-BCMA/anti-CD3 T-cell bispecific antibodies as measured by flowcytometry. Concentration-dependent killing of JJN-3 MM cells by 22-TCBcv(closed triangle), 42-TCBcv (closed square) in comparison with83A10-TCBcv (open circle, dotted line). Percentage of annexin-V positiveJJN-3 cells (FIG. 7A, FIG. 7C) and tumor cell lysis (FIG. 7B, FIG. 7D)were determined and plotted. The percentage of lysis of JJN-3 cellsinduced by a specific concentration of anti-BCMA/anti-CD3 T cellbispecific antibody determined as the following: the absolute count ofannexin-V-negative JJN-3 cells at a given TCB concentration andsubtracting it from the absolute count of annexin-V-negative JJN-3 cellswithout TCB; divided by the absolute count of annexin-V-negative JJN-3cells without TCB. Experiments were performed with 2 PBMC donors: donor1 (FIG. 7A, FIG. 7B) and donor 2 (FIG. 7C, FIG. 7D) using an E:T ratioof 10 PBMCs to 1 MM cell (see example 11).

FIGS. 8A-8C. Redirected T-cell lysis of multiple myeloma patient bonemarrow myeloma plasma cells in presence of autologous bone marrowinfiltrating T cells (patient's whole bone marrow aspirates) induced byanti-BCMA/anti-CD3 T-cell bispecific antibodies as measured bymultiparameter flow cytometry. Percentage of annexin-V positive myelomaplasma cells was determined and plotted against TCB concentrations.Concentration-dependent and specific lysis of patient myeloma plasmacells were observed while lysis of T cells, B cells, and NK cells wasnot observed based on an 8-color multiparameter panel. No induction ofcell death of myeloma plasma cells with control-TCB at the highestconcentration of TCB antibodies tested. As compared to 83A10-TCBcv (FIG.8A), 42-TCBcv (FIG. 8B) and 22-TCBcv (FIG. 8C) were more potent toinduce killing of patient bone marrow myeloma plasma cells (see example13).

FIGS. 9A-9B. Redirected T-cell lysis of multiple myeloma patient bonemarrow myeloma plasma cells in presence of autologous bone marrowinfiltrating T cells (patient's whole bone marrow aspirates) induced byanti-BCMA/anti-CD3 T-cell bispecific antibodies as measured by flowcytometry. Percentage of annexin-V negative myeloma plasma cells wasdetermined and plotted against TCB concentrations.Concentration-dependent and specific lysis of patient myeloma plasmacells were observed while lysis of non-malignant bone marrow cells wasnot observed (data not shown). No induction of cell death of myelomaplasma cells observed with control-TCB at the highest concentration ofTCB antibodies tested (data not shown). As compared to 83A10-TCBcv,42-TCBcv and 22-TCBcv were more potent to induce killing of patient bonemarrow myeloma plasma cells as reflected by the concentration-dependentreduction of viable (annexin-V negative) myeloma plasma cells.Representative experiments in patient 001 (FIG. 9A) and patient 007(FIG. 9B) (see example 13).

FIGS. 10A-10H. Redirected T-cell lysis of multiple myeloma patient bonemarrow myeloma plasma cells in presence of autologous bone marrowinfiltrating T cells induced by anti-BCMA/anti-CD3 T-cell bispecificantibodies as measured by flow cytometry. Percentage of propidium iodidenegative myeloma plasma cells was determined and the percentage ofviable bone marrow plasma cells relative to the medium control (MC) wasplotted against TCB concentrations. Concentration-dependent and specificlysis of patient myeloma plasma cells were observed (FIG. 10A-FIG. 10G)while lysis of bone marrow microenvironment (BMME) was not observed(FIG. 10H). No induction of cell death of myeloma plasma cells observedwith control-TCB at the highest concentration of TCB antibodies tested.As compared to 83A10-TCBcv, 42-TCBcv and 22-TCBcv were more potent toinduce killing of patient bone marrow myeloma plasma cells as reflectedby the concentration-dependent reduction of viable (propidium iodidenegative) myeloma plasma cells. An effect was considered statisticallysignificant if the P-value of its corresponding statistical test was <5%(*), <1% (**) or <0.1% (***). Experiments performed using bone marrowaspirate samples collected from patient 1 (FIG. 10A), patient 2 (FIG.10B), patient 3 (FIG. 10C), patient 4 (FIG. 10D), patient 5 (FIG. 10E),patient 6 (FIG. 10F), and patient 7 (FIG. 10G, FIG. 10H) (see example13).

FIGS. 11A-11C. Activation of myeloma patient bone marrow T cells inpresence of bone marrow plasma cells (patient whole bone marrowaspirates) induced by anti-BCMA/anti-CD3 T-cell bispecific antibodies asmeasured by multiparameter flow cytometry (8-color staining panel). FIG.11A: Magnitude of T-cell activation was shown for 83A10-TCBcv. FIG. 11B:Magnitude of T-cell activation (top graph: CD4 T-Cell Activation; bottomgraph: CD8 T-Cell Activation) was shown for 42-TCBcv. FIG. 11C:Magnitude of T-cell activation (top graph: CD4 T-Cell Activation; bottomgraph: CD8 T-Cell Activation) was shown for 22-TCBcv (see example 14).

FIG. 12. Concentrations of 83A10-TCBcv measured from serum samples(closed symbols with full lines) and bone marrow samples (open symbolswith dotted lines) after single intravenous (IV) injection in cynomolgusmonkeys with 0.003, 0.03 and 0.1 mg/kg of 83A10-TCBcv. Serum samplescollection was performed at pre-dose and 30, 90, 180 min, 7, 24, 48, 96,168, 336, 504 h after dosing. Bone marrow samples were collected atpre-dose, and 96 and 336 h after dosing (see example 16).

FIG. 13. Peripheral T-cell redistribution observed in cynomolgus monkeysfollowing a single IV injection of 83A10-TCBcv (0.003, 0.03 and 0.3mg/kg) Animals A and B, C and D, and E and F respectively received an IVinjection of 0.003, 0.03 and 0.3 mg/kg of 83A10-TCBcv. Absolute bloodT-cell cell counts (CD2+ cells per μL of blood) were plotted againsttime post treatment (see example 16).

FIGS. 14A-14B. Reduction of blood plasma cells observed in cynomolgusmonkeys following a single IV injection of 83A10-TCBcv (0.3 mg/kg) asmeasured by multiparameter flow cytometry. FIG. 14A: Plasma cells (PCs)were identified based on a 6-color staining panel and percentages of PCsover lymphocytes were measured and plotted in contour plots. FIG. 14B:Kinetic of blood plasma cell depletion after treatment with 83A10-TCBcv0.3 mg/kg in cynomolgus monkeys was plotted (see example 16).

FIGS. 15A-15C. Antitumoral activity induced by 83A10-TCBcvanti-BCMA/anti-CD3 T cell bispecific antibody in the H929 human myelomaxenograft model using PBMC-humanized NOG mice. ImmunodeficientNOD/Shi-scid IL2rgamma(null) (NOG) received on day 0 (d0) human multiplemyeloma H929 cells as a subcutaneous (SC) injection into the rightdorsal flank. On day 15 (d15), NOG mice received a singleintraperitoneal (IP) injection of human PBMCs. Mice were then carefullyrandomized into the different treatment and control groups (n=9/group)and a statistical test was performed to test for homogeneity betweengroups. The experimental groups were the control untreated group,control-TCB treated group, 83A10-TCBcv 2.6 nM/kg treated group andBCMA50-BiTE® (BCMA×CD3 (scFv)₂) 2.6 nM/kg treated group. Antibodytreatment given by tail vein injection started on day 19 (d19), i.e. 19days after SC injection of H929 tumor cells. The TCB antibody treatmentschedule consisted of a once a week IV administration for up to 3 weeks(i.e. total of 3 injections of TCB antibody). Tumor volume (TV) wasmeasured by caliper during the study and progress evaluated byintergroup comparison of TV. TV (mm3) plotted against day post tumorinjection. On d19, first day of treatment, the mean tumor volume hadreached 300±161 mm3 for the vehicle treated control group (FIG. 15A),315±148 mm3 for the 2.6 nM/kg control-TCB treated group (FIG. 15A),293±135 mm3 for the 2.6 nM/kg 83A10-TCBcv group (FIG. 15B) and 307±138mm3 for the 2.6 nM/kg BCMA50-BiTE® group (FIG. 15C). TV of eachindividual mouse per experimental group were plotted against day posttumor injection: (FIG. 15A) control groups including vehicle control(full line) and control-TCB (dotted line), (FIG. 15B) 83A10-TCBcv (2.6nM/kg) group, and (FIG. 15C) BCMA50-BiTE® (2.6 nM/kg). Black arrows showthe TCB treatment given by IV injection. In the 83A10-TCBcv (2.6 nM/kg)group, 6 out of 9 mice (67%) had their tumor regressed even below TVrecorded at d19 i.e. first TCB treatment and tumor regression wasmaintained until termination of study. The 3 mice in the 83A10-TCBcv(2.6 nM/kg) treated group which failed to show tumor regression hadtheir TV equal to 376, 402 and 522 mm3 respectively at d19. In contrast,none of the 9 mice (0%) treated with an equimolar dose of BCMA50-BiTE®(2.6 nM/kg) at a once a week schedule for 3 weeks had their tumorregressed at any timepoint (see example 17).

FIG. 16. Percentage of tumor growth (TG) calculated for d19 to d43 andcompared between 83A10-TCBcv (2.6 nM/kg) group and BCMA50-BiTE® (2.6nM/kg). The percentage of tumor growth defined as TG (%) was determinedby calculating TG (%)=100×(median TV of analyzed group)/(median TV ofcontrol vehicle treated group). For ethical reason, mice were euthanizedwhen TV reached at least 2000 mm3 TG (%) was consistently andsignificantly reduced in the 83A10-TCBcv (2.6 nM/kg) group as well asthe TG (%) was always lower when compared to BCMA50-BiTE® (2.6 nM/kg)(see example 17).

FIGS. 17A-17B. Surface plasmon resonance (SPR) of 70 clones selectedfrom ELISA. All experiments were performed at 25° C. using PBST asrunning buffer (10 mM PBS, pH 7.4 and 0.005% (v/v) Tween®20) with aProteOn XPR36 biosensor equipped with GLC and GLM sensor chips andcoupling reagents. Immobilizations were performed at 30 μl/min on a GLMchip. pAb (goat) anti hu IgG, F(ab)2 specific Ab (Jackson) was coupledin vertical direction using a standard amine-coupling procedure: all sixligand channels were activated for 5 min with a mixture of EDC (200 mM)and sulfo-NHS (50 mM) Immediately after the surfaces were activated, pAb(goat) anti hu IgG, F(ab)2 specific antibody (50 μg/ml, 10 mM sodiumacetate, pH 5) was injected across all six channels for 5 min. Finally,channels were blocked with a 5 min injection of 1 M ethanolamine-HCl (pH8.5). Final immobilization levels were similar on all channels, rangingfrom 11000 to 11500 RU. The Fab variants were captured from E.colisupernatants by simultaneous injection along five of the separate wholehorizontal channels (30 μl/min) for 5 min and resulted in levels,ranging from 200 to 900 RU, depending on the concentration of Fab insupernatant; conditioned medium was injected along the sixth channel toprovide an ‘in-line’ blank for double referencing purposes. FIG. 17A:One-shot kinetic measurements were performed by injection of a dilutionseries of human (50, 10, 2, 0.4, 0.08, 0 nM, 50 μl/min) for 3 min alongthe vertical channels. Dissociation was monitored for 5 min. Kineticdata were analyzed in ProteOn Manager v. 2.1. FIG. 17B: One-shot kineticmeasurements were performed by injection of a dilution series of cynoBCMA (50, 10, 2, 0.4, 0.08, 0 nM, 50 μl/min) for 3 min along thevertical channels. Dissociation was monitored for 5 min. Kinetic datawere analyzed in ProteOn Manager v. 2.1. Processing of the reaction spotdata involved applying an interspot-reference and a double-referencestep using an inline buffer blank (Myszka, 1999). The processed datafrom replicate one-shot injections were fit to a simple 1:1 Langmuirbinding model without mass transport (O'Shannessy et al., 1993).

FIGS. 18A-18B. FIG. 18A: Binding affinity of BCMA antibodies onHEK-huBCMA cells as measured by flow cytometry. The anti-BCMA antibodieswere used as first antibody then a secondary PE-labeled anti-human Fcwas used as detection antibody. It was found that binding of antibodiesMab 21, Mab 22, Mab 27, Mab 39 and Mab 42 to huBCMA on HEK cells was notsignificantly better than the binding of Mab 83A10 to huBCMA-HEK cells.FIG. 18B: Binding affinity of BCMA antibodies on HEK-huBCMA cells asmeasured by flow cytometry. The anti-BCMA antibodies were used as firstantibody then a secondary PE-labeled anti-human Fc was used as detectionantibody. It was found that binding of antibodies Mab 21, Mab 22, Mab27, Mab 39 and Mab 42 to huBCMA on HEK cells was not significantlybetter than the binding of Mab 83A10 to huBCMA-HEK cells.

FIGS. 19A-19B. Concentrations of 42-TCBcv measured in serum and bonemarrow after single IV or SC injection in cynomolgus monkeys. Animalsreceived a single IV (FIG. 19A) or SC (FIG. 19B) injection of 42-TCBcvand blood samples per timepoint were collected via the peripheral veinfor PK evaluations at Pre-dose, 30, 90, 180 min, 7, 24, 48, 96, 168,336, 504 h after dosing. Blood samples were allowed to clot in tubes forserum separation for 60 min at room temperature. The clot was spun downby centrifugation. The resultant serum was directly stored at −80° C.until further analysis. Bone marrow samples for PK evaluations were alsocollected at the femur under anesthesia/analgesic treatment at Pre-dose,96 and 336 h after dosing. Bone marrow samples were allowed to clot intubes for serum separation for 60 min at room temperature. The clot wasspun down by centrifugation. The resultant bone marrow was directlystored at −80° C. until further analysis. The PK data analysis andevaluation were performed. Standard non compartmental analysis wasperformed using Watson package (v 7.4, Thermo Fisher Scientific Waltman,Mass., USA) or Phoenix WinNonlin system (v. 6.3, Certara Company, USA).Effective concentration range of 42-TCBcv in multiple myeloma patientbone marrow aspirates corresponding to 10 μm to 10 nM (grey area).Concentrations in parenthesis are in nM.

FIGS. 20A-20B. Redirected T-cell lysis of plasma cell leukemia patientbone marrow leukemic cells in presence of autologous T cells or bonemarrow infiltrating T cells induced by anti-BCMA/anti-CD3 T-cellbispecific antibodies as measured by flow cytometry. Percentage ofpropidium iodide negative myeloma plasma cells was determined and thepercentage of viable bone marrow plasma cell leukemic cells relative tothe medium control (MC) was plotted against TCB concentrations.Concentration-dependent and specific lysis of patient plasma cellleukemic cells were observed (FIG. 20A, FIG. 20B) while lysis of bonemarrow microenvironment (BMME) was not observed (data not shown). Noinduction of cell death of myeloma plasma cells observed withcontrol-TCB at the highest concentration of TCB antibodies tested.42-TCBcv was very potent to induce killing of patient bone marrow plasmacell leukemic cells as reflected by the concentration-dependentreduction of viable (propidium iodide negative) myeloma plasma cells. Aneffect was considered statistically significant if the P-value of itscorresponding statistical test was <5% (*), <1% (**) or <0.1% (***). Thefigure shows results obtained from bone marrow samples of patient 1(FIG.20A) and patient 2(FIG. 20B) (see also example 20).

FIGS. 21A-21B. Redirected T-cell lysis of H929 MM cells induced byanti-BCMA/anti-CD3 T-cell bispecific antibodies in combination withthalidomide derivatives (lenalidomide) or immunotherapeutic derivatives(anti-PD1 and anti-CD38 antibodies) as measured by flow cytometry. H929MM cells were co-cultured with human leukocytes (n=1 or 5) andchallenged to suboptimal concentrations of anti-BCMA/anti-CD3 T-cellbispecific antibody A) 83A10-TCcv (10 pM) (FIG. 21A) or 42-TCBcv (10 pM)(FIG. 21B) alone, in combination with suboptimal concentrations oflenalidomide (1 μM), anti-PD-1 (10 μg/ml) and anti-CD38 daratumumab (10μg/ml). Combining anti-BCMA/anti-CD3 T-cell bispecific antibody83A10-TCBcv (n=5) with lenalidomide or anti-CD38 daratumumabsignificantly increased their anti-MM efficacy by 4-fold and 2.5-fold,respectively (FIG. 21A). Combining 42-TCBcv (n=1) with lenalidomide ordaratumumab also increased their anti-MM efficacy to kill MM cell lines(FIG. 21B).

DETAILED DESCRIPTION OF THE INVENTION

The term “BCMA, the target BCMA, human BCMA” as used herein relates tohuman B cell maturation antigen, also known as BCMA; TR17_HUMAN,TNFRSF17 (UniProt Q02223), which is a member of the tumor necrosisreceptor superfamily that is preferentially expressed in differentiatedplasma cells. The extracellular domain of BCMA consists according toUniProt of amino acids 1-54 (or 5-51). The term “antibody against BCMA,anti-BCMA antibody” as used herein relates to an antibody specificallybinding to the extracellular domain of BCMA.

“Specifically binding to BCMA or binding to BCMA” refer to an antibodythat is capable of binding to the target BCMA with sufficient affinitysuch that the antibody is useful as a therapeutic agent in targetingBCMA. In some embodiments, the extent of binding of an anti-BCMAantibody to an unrelated, non-BCMA protein is about 10-foldpreferably >100-fold less than the binding of the antibody to BCMA asmeasured, e.g., by surface plasmon resonance (SPR) e.g. Biacore®,enzyme-linked immunosorbent (ELISA) or flow cytometry (FACS). In oneembodiment the antibody that binds to BCMA has a dissociation constant(Kd) of 10⁻⁸ M or less, preferably from 10⁻⁸ M to 10⁻¹³ M, preferablyfrom 10⁻⁹ M to 10⁻¹³ M. In one embodiment the anti-BCMA antibody bindsto an epitope of BCMA that is conserved among BCMA from differentspecies, preferably among human and cynomolgus, and in additionpreferably also to mouse and rat BCMA. “Bispecific antibody specificallybinding to CD3 and BCMA, bispecific antibody against CD3 and BCMA”refers to a respective definition for binding to both targets. Anantibody specifically binding to BCMA (or BCMA and CD3) does not bind toother human antigens. Therefore in an ELISA, OD values for suchunrelated targets will be equal or lower to that of the limit ofdetection of the specific assay, preferably >0.3 ng/mL, or equal orlower to OD values of control samples without plate-bound-BCMA or withuntransfected HEK293 cells.

Preferably the anti-BCMA antibody is specifically binding to a group ofBCMA, consisting of human BCMA and BCMA of non-human mammalian origin,preferably BCMA from cynomolgus, mouse and/or rat. “cyno/human gap”refer to the affinity ratio KD cynomolgus BCMA [M]/KD human BCMA [M](details see example 3). “cyno/human gap of Mab CD3” as used hereinrefer to affinity ratio KD cynomolgus CD3 [M]/KD human CD3 [M]. In oneembodiment the bispecific anti-BCMA/anti-CD3 antibody of the inventionshows a cyno/human gap of Mab CD3 between 1.25 and 5 or between 0.8 and1.0. The bispecific antibody according to the invention is in oneembodiment characterized in that it binds also specifically tocynomolgus CD3. In one embodiment the bispecific anti-BCMA/anti-CD3antibody of the invention shows a cyno/human gap of Mab CD3 between 1.25and 5 or between 0.8 and 1.0. Preferably the cyno/human gap is in thesame range for anti-BCMA- and the anti-CD3 antibody.

The term “APRIL” as used herein relates to recombinant, truncated murineAPRIL (amino acids 106-241; NP_076006). APRIL can be produced asdescribed in Ryan, 2007 (Mol Cancer Ther; 6 (11): 3009-18).

The term “BAFF” as used herein relates to recombinant, truncated humanBAFF (UniProt Q9Y275 (TN13B_HUMAN) which can be produced as described inGordon, 2003 (Biochemistry; 42 (20): 5977-5983). Preferably a His-taggedBAFF is used according to the invention. Preferably the His-tagged BAFFis produced by cloning a DNA fragment encoding BAFF residues 82-285 intoan expression vector, creating a fusion with an N-terminal His-tagfollowed by a thrombin cleavage site, expressing said vector andcleaving the recovered protein with thrombin.

Anti-BCMA antibodies are analyzed by ELISA for binding to human BCMAusing plate-bound BCMA. For this assay, an amount of plate-bound BCMApreferably 1.5 μg/mL and concentration(s) ranging from 0.1 pM to 200 nMof anti-BCMA antibody are used.

The term “NF-κB” as used herein relates to recombinant NF-κB p50(accession number (P19838). NF-κB activity can be measured by aDNA-binding ELISA of an extract of NCI-H929 MM cells (CRL-9068™).NCI-H929 MM cells, untreated or treated with 0.1 μg/mL TNF-α, 1000 ng/mLheat-treated HT-truncated-BAFF, 1000 ng/mL truncated-BAFF, 0.1 pM to 200nM isotype control, and with or without 0.1 pM to 200 nM anti-BCMAantibodies are incubated for 20 min. NF-κB activity can be assayed usinga functional ELISA that detects chemiluminescent signal from p65 boundto the NF-κB consensus sequence (U.S. Pat. No. 6,150,090).

The term “further target” as used herein means preferably CD3c. The term“first target and second target” means either CD3 as first target andBCMA as second target or means BCMA as first target and CD3 as secondtarget.

The term “CD3ε or CD3” as used herein relates to human CD3ε describedunder UniProt P07766 (CD3E_HUMAN). The term “antibody against CD3ε, antiCD3ε antibody” relates to an antibody specifically binding to CD3ε. Inone embodiment the antibody comprises a variable domain VH comprisingthe heavy chain CDRs of SEQ ID NO: 1, 2 and 3 as respectively heavychain CDR1H, CDR2H and CDR3H and a variable domain VL comprising thelight chain CDRs of SEQ ID NO: 4, 5 and 6 as respectively light chainCDR1L, CDR2L and CDR3L. In one embodiment the antibody comprises thevariable domains of SEQ ID NO:7 (VH) and SEQ ID NO:8 (VL).

The term “anti-CD38 antibody” as used herein relates to an antibodyspecifically binding to human CD38. In an embodiment of the inventionthe anti-CD38 antibody is daratumumab (US20150246123). In an embodimentof the invention the anti-CD38 antibody is isatuximab (SAR650984, U.S.Pat. No. 8,877,899). In an embodiment of the invention the anti-CD38antibody is MOR202 (WO 2012041800). In an embodiment of the inventionthe anti-CD38 antibody is Ab79 (U.S. Pat. No. 8,362,211). In anembodiment of the invention the anti-CD38 antibody is Ab19 (U.S. Pat.No. 8,362,211). The dosage of such anti-CD38 antibody is performedaccording to the state of the art and described in the respectiveprescribing informations. E.g. Daratumumab dosage is usually 16 mg/kg(www.ema.europa.eu).

The term “thalidomide compound” or “thalidomide and an immunotherapeuticderivative” as used herein relates to2-(2,6-dioxopiperidin-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione andimmunotherapeutic derivatives thereof. In an embodiment of the inventionthe thalidomide compound is selected from the group consisting of, butnot limited to, thalidomide (CAS Registry Number 50-35-1), lenalidomide(CAS Registry Number 191732-72-6), pomalidomide (CAS Registry Number19171-19-8), CC122 (CAS Registry Number 1398053-45-6) and CC-220 (CASRegistry Number 1323403-33-3) and the respective salts (preferably HClsalts 1:1). The chemical formula of CC-122 is2,6-piperidinedione,3-(5-amino-2-methyl-4-oxo-3(4H-quinazolinyl),hydrochloride (1:1) and of CC-220 it is 2,6-piperidinedione,3-[1,3-dihydro-4-[[4-(4-morpholinylmethyl)phenyl]methoxy]-1-oxo-2H-isoindol-2-yl]-,(3S)-, hydrochloride (1:1). Methods of preparing CC-220 are described,e.g., in US 20110196150, the entirety of which is incorporated herein byreference.

The dosage of thalomide compounds is performed according to the state ofthe art and described in the respective prescribing informations. E.g.Revlimid® (lenalidomide) dosage is usually 25 mg once daily orally ondays 1-21 of repeated 28-day cycles (www.revlimid.com) and POMALYST®(pomalidomide) dosage for the treatment of Multiple Myeloma is usually 4mg per day taken orally on days 1-21 of repeated 28-day cycles(www.celgene.com). In one embodiment,3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione isadministered in an amount of about 5 to about 50 mg per day.

In one embodiment, CC-122 and CC-220 are administered in an amount ofabout 5 to about 25 mg per day. In another embodiment, CC-122 and CC-220are administered in an amount of about 5, 10, 15, 25, 30 or 50 mg perday. In another embodiment, 10 or 25 mg of CC-122 and CC-220 areadministered per day. In one embodiment, CC-122 and CC-220 areadministered twice per day.

The term “anti-PD-1 antibody” as used herein relates to an antibodyspecifically binding to human PD-1. Such antibodies are e.g. describedin WO2015026634 (MK-3475, pembrolizumab), U.S. Pat. Nos. 7,521,051,5,800,8449, and 8,354,509. Pembrolizumab (Keytruda®, MK-3475.) is alsodescribed in WO 2009/114335, Poole, R. M. Drugs (2014) 74: 1973;Seiwert, T., et al., J. Clin. Oncol. 32,5s (suppl; abstr 6011). In anembodiment of the invention the PD-1 antibody is MK-3475 (WHO DrugInformation, Vol. 27, No. 2, pages 161-162 (2013)) and which comprisesthe heavy and light chain amino acid sequences shown in FIG. 6 of WO2015026634 The amino acid sequence of pembrolizumab is described inWO2008156712 (light chain CDRs SEQ ID NOS:15, 16 and 17 and heavy chainCDRs SEQ ID NOS: 18, 19 and 20). In an embodiment of the invention thePD-1 antibody is nivolumab (BMS-936558, MDX 1106; WHO Drug Information,Vol. 27, No. 1, pages 68-69 (2013), WO2006/121168 amino acid sequencesshown in WO 2015026634). In an embodiment of the invention the PD-1antibody is; pidilizumab (CT-011, also known as hBAT or hBAT-1; aminoacid sequence see WO2003/099196; WO 2009/101611, Fried I. et al.; NeuroOncol (2014) 16 (suppl 5): viii-v112). In an embodiment of the inventionthe PD-1 antibody is MEDI-0680 (AMP-514, WO2010/027423, WO2010/027827,WO2010/027828, Hamid 0. et al.; J Clin Oncol 33, 2015 (suppl; abstrTPS3087). In an embodiment of the invention the PD-1 antibody is PDR001(Naing A. et al.; J Clin Oncol 34, 2016 (suppl; abstr 3060). In anembodiment of the invention the PD-1 antibody is REGN2810 (PapadopoulosK P et al.; J Clin Oncol 34, 2016 (suppl; abstr 3024). In an embodimentof the invention the PD-1 antibody is lambrolizumab (WO2008/156712). Inan embodiment of the invention the PD-1 antibody is h409A1 1, h409A16 orh409A17, which are described in WO2008/156712. The dosage of suchanti-PD-1 antibody is performed according to the state of the art anddescribed in the respective prescribing informations. E.g. Keytruda® isadministered usually in a concentration of 2 mg/kg body weight everythree weeks (http://ec.europa.eu/health/documents).

The term “anti-PD-L1 antibody” as used herein relates to an antibodyspecifically binding to human PD-L1. Such antibodies are e.g. describedin WO2015026634, WO2013/019906, WO2010/077634 and U.S. Pat. No.8,383,796. In an embodiment of the invention the PD-L1 antibody isMPDL3280A (atezolizumab, YW243.55.570, WO2010/077634, McDermott D F. Etal., JCO Mar. 10, 2016 vol. 34 no. 8 833-842). In an embodiment of theinvention the PD-L1 antibody is MDX-1105 (BMS-936559, WO2007/005874,Patrick A. Ott P A et al., DOI: 10.1158/1078-0432, Clinical CancerResearch-13-0143). In an embodiment of the invention the PD-L1 antibodyis MEDI4736 (durvalumab, WO 2016/040238 Gilbert J. et al., Journal forImmunoTherapy of Cancer 20153(Suppl 2):P152). In an embodiment of theinvention the PD-L1 antibody is MSB001071 8C (avelumab, Disis M L. etal., Journal of Clinical Oncology, Vol 33, No 15_suppl (May 20Supplement), 2015: 5509). In an embodiment of the invention the PD-L1antibody is the anti-PD-L1 antibody comprising a VH sequence of SEQ IDNO: 16 and a VL sequence of SEQ ID NO: 17 as described in WO2016007235.The dosage of such anti-PD-L1 antibody is performed according to thestate of the art and described in the respective prescribinginformations. E.g. atezolizumab is administered usually in aconcentration of 1200 mg as an intravenous infusion over 60 minutesevery 3 weeks (www.accessdata.fda.gov).

The term “antibody” as used herein refers to a monoclonal antibody. Anantibody consists of two pairs of a “light chain” (LC) and a “heavychain” (HC) (such light chain (LC)/heavy chain pairs are abbreviatedherein as LC/HC). The light chains and heavy chains of such antibodiesare polypeptides consisting of several domains. Each heavy chaincomprises a heavy chain variable region (abbreviated herein as HCVR orVH) and a heavy chain constant region. The heavy chain constant regioncomprises the heavy chain constant domains CH1, CH2 and CH3 (antibodyclasses IgA, IgD, and IgG) and optionally the heavy chain constantdomain CH4 (antibody classes IgE and IgM). Each light chain comprises alight chain variable domain VL and a light chain constant domain CL. Thevariable domains VH and VL can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each VH and VL is composed of three CDRs and four FRs,arranged from amino-terminus to carboxy-terminus in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The “constant domains” of theheavy chain and of the light chain are not involved directly in bindingof an antibody to a target, but exhibit various effector functions. Theterm “antibody” as used herein refers comprises also the portion of anantibody which is needed at least for specific binding to the antigenCD3 resp. BCMA. Therefore such an antibody (or antibody portion) can bein one embodiment a Fab fragment, if said antibody portion is comprisedin a bispecific antibody according to the invention. The antibodyaccording to the invention can also be a Fab′, F(ab′)₂, a scFv, adi-scFv, or a bi-specific T-cell engager (BiTE).

The term “antibody” includes e.g. mouse antibodies, human antibodies,chimeric antibodies, humanized antibodies and genetically engineeredantibodies (variant or mutant antibodies) as long as theircharacteristic properties are retained. Especially preferred are humanor humanized antibodies, especially as recombinant human or humanizedantibodies. Further embodiments are heterospecific antibodies(bispecific, trispecific etc.) and other conjugates, e.g. with cytotoxicsmall molecules.

The term “bispecific antibody” as used herein refers in one embodimentto an antibody in which one of the two pairs of heavy chain and lightchain (HC/LC) is specifically binding to CD3 and the other one isspecifically binding to BCMA. The term also refers to other formats ofbispecific antibodies according to the state of the art, in oneembodiment to bispecific single-chain antibodies.

The term “TCB” as used herein refer to a bispecific antibodyspecifically binding to BCMA and CD3. The term “83A10-TCBcv” as usedherein refer to a bispecific antibody specifically binding to BCMA andCD3 as specified by its heavy and light chain combination of SEQ IDNO:45, SEQ ID NO:46, SEQ ID NO:47 (2×), and SEQ ID NO:48, and as shownin FIG. 2A and described in EP14179705. The terms “21-TCBcv, 22-TCBcv,42-TCBcv” as used herein refer to the respective bispecific antibodiesof Mab21, as specified by its heavy and light chain combination of SEQID NO:48, SEQ ID NO:49, SEQ ID NO:50, and SEQ ID NO:51 (2×), Mab 22 asspecified by its heavy and light chain combinations of SEQ ID NO:48, SEQID NO:52, SEQ ID NO:53, and SEQ ID NO:54 (2×), and Mab42 as specified byits heavy and light chain combination of SEQ ID NO:48 of SEQ ID NO:55,SEQ ID NO:56, and SEQ ID NO:57-(2×).

The term “naked antibody” as used herein refers to an antibody which isspecifically binding to BCMA, comprising an Fc part and is notconjugated with a therapeutic agent e.g. with a cytotoxic agent orradiolabel. The term “conjugated antibody, drug conjugate” as usedherein refers to an antibody which is specifically binding to BCMA, andis conjugated with a therapeutic agent e.g. with a cytotoxic agent orradiolabel.

The term “bispecific single-chain antibody” as used herein refers to asingle polypeptide chain comprising in one embodiment two bindingdomains, one specifically binding to BCMA and the other one in oneembodiment specifically binding to CD3. Each binding domain comprisesone variable region from an antibody heavy chain (“VH region”), whereinthe VH region of the first binding domain specifically binds to the CD3molecule, and the VH region of the second binding domain specificallybinds to BCMA. The two binding domains are optionally linked to oneanother by a short polypeptide spacer. A non-limiting example for apolypeptide spacer is Gly-Gly-Gly-Gly-Ser (G-G-G-G-S) and repeatsthereof. Each binding domain may additionally comprise one variableregion from an antibody light chain (“VL region”), the VH region and VLregion within each of the first and second binding domains being linkedto one another via a polypeptide linker, long enough to allow the VHregion and VL region of the first binding domain and the VH region andVL region of the second binding domain to pair with one another suchthat, together, they are able to specifically bind to the respectivefirst and second binding domains (see e.g. EP0623679).Bispecificsingle-chain antibodies are also mentioned e.g. in Choi B D et al.,Expert Opin Biol Ther. 2011 July; 11(7):843-53 and Wolf E. et al., DrugDiscov Today. 2005 Sep. 15; 10(18):1237-44.

The term “diabody” as used herein refers to a small bivalent andbispecific antibody fragment comprising a heavy (VH) chain variabledomain connected to a light chain variable domain (VL) on the samepolypeptide chain (VH-VL) connected by a peptide linker that is tooshort to allow pairing between the two domains on the same chain(Kipriyanov, Int. J. Cancer 77 (1998), 763-772). This forces pairingwith the complementary domains of another chain and promotes theassembly of a dimeric molecule with two functional antigen bindingsites. To construct bispecific diabodies of the invention, the V-domainsof an anti-CD3 antibody and an anti-BCMA antibody are fused to createthe two chains VH(CD3)-VL(BCMA), VH(BCMA)-VL(CD3). Each chain by itselfis not able to bind to the respective antigen, but recreates thefunctional antigen binding sites of anti-CD3 antibody and anti-BCMAantibody on pairing with the other chain. The two scFv molecules, with alinker between heavy chain variable domain and light chain variabledomain that is too short for intramolecular dimerization, areco-expressed and self-assemble to form bi-specific molecules with thetwo binding sites at opposite ends. By way of example, the variableregions encoding the binding domains for BCMA and CD3, respectively, canbe amplified by PCR from DNA constructs obtained as described, such thatthey can be cloned into a vector like pHOG, as described in Kipiriyanovet al., J. Immunol, Methods, 200, 69-77 (1997a). The two scFV constructsare then combined in one expression vector in the desired orientation,whereby the VH-VL linker is shortened to prevent backfolding of thechains onto themselves. The DNA segments are separated by a STOP codonand a ribosome binding site (RBS). The RBS allows for the transcriptionof the mRNA as a bi-cistronic message, which is translated by ribosomesinto two proteins which non-covalently interact to form the diabodymolecule. Diabodies, like other antibody fragments, have the advantagethat they can be expressed in bacteria (E. coli) and yeast (Pichiapastoris) in functional form and with high yields (up to Ig/l).

The term “tandem scFVs” as used herein refers to a single chain Fvmolecule (i.e. a molecule formed by association of the immunoglobulinheavy and light chain variable domains, VH and VL, respectively) asdescribed e.g., in WO 03/025018 and WO 03/048209. Such Fv molecules,which are known as TandAbs® comprise four antibody variable domains,wherein (i) either the first two or the last two of the four variabledomains bind intramolecularly to one another within the same chain byforming an antigen binding scFv in the orientation VH/VL or VL/VH (ii)the other two domains bind intermolecularly with the corresponding VH orVL domains of another chain to form antigen binding VH/VL pairs. In apreferred embodiment, as mentioned in WO 03/025018, the monomers of suchFv molecule comprise at least four variable domains of which twoneighboring domains of one monomer form an antigen-binding VH-VL orVL-VH scFv unit.

The term “DARPins” as used herein refers to a bispecific ankyrin repeatmolecule as described e.g. in US 2009082274. These molecules are derivedfrom natural ankyrin proteins, which can be found in the human genomeand are one of the most abundant types of binding proteins. A DARPinlibrary module is defined by natural ankyrin repeat protein sequences,using 229 ankyrin repeats for the initial design and another 2200 forsubsequent refinement. The modules serve as building blocks for theDARPin libraries. The library modules resemble human genome sequences. ADARPin is composed of 4 to 6 modules. Because each module is approx. 3.5kDa, the size of an average DARPin is 16-21 kDa. Selection of binders isdone by ribosome display, which is completely cell-free and is describedin He M and Taussig M J., Biochem Soc Trans. 2007, November; 35(Pt5):962-5.

The term “T cell bispecific engager” are fusion proteins consisting oftwo single-chain variable fragments (scFvs) of different antibodies, oramino acid sequences from four different genes, on a single peptidechain of about 55 kilodaltons. One of the scFvs binds to T cells via theCD3 receptor, and the other to a BCMA.

There are five types of mammalian antibody heavy chains denoted by theGreek letters: α, δ, ε, γ, and μ (Janeway C A, Jr et al (2001).Immunobiology. 5th ed., Garland Publishing). The type of heavy chainpresent defines the class of antibody; these chains are found in IgA,IgD, IgE, IgG, and IgM antibodies, respectively (Rhoades R A, Pflanzer RG (2002). Human Physiology, 4th ed., Thomson Learning). Distinct heavychains differ in size and composition; α and γ contain approximately 450amino acids, while μ and ε have approximately 550 amino acids.

Each heavy chain has two regions, the constant region and the variableregion. The constant region is identical in all antibodies of the sameisotype, but differs in antibodies of different isotype. Heavy chains γ,α and δ have a constant region composed of three constant domains CH1,CH2, and CH3 (in a line), and a hinge region for added flexibility (WoofJ, Burton D Nat Rev Immunol 4 (2004) 89-99); heavy chains μ and ε have aconstant region composed of four constant domains CH1, CH2, CH3, and CH4(Janeway C A, Jr et al (2001). Immunobiology. 5th ed., GarlandPublishing). The variable region of the heavy chain differs inantibodies produced by different B cells, but is the same for allantibodies produced by a single B cell or B cell clone. The variableregion of each heavy chain is approximately 110 amino acids long and iscomposed of a single antibody domain.

In mammals there are only two types of light chain, which are calledlambda (λ) and kappa (κ). A light chain has two successive domains: oneconstant domain CL and one variable domain VL. The approximate length ofa light chain is 211 to 217 amino acids. In one embodiment the lightchain is a kappa (κ) light chain, and the constant domain CL is in oneembodiment derived from a kappa (K) light chain (the constant domainCK).

“aa substitution” as used herein refer to independent amino acidsubstitution in the constant domain CH1 at the amino acid at positions147 and 213 by glutamic acid (E), or aspartic acid (D) and in theconstant domain CL the amino acid at position 124 is substituted bylysine (K), arginine (R) or histidine (H). In one embodiment in additionin the constant domain CL the amino acid at position 123 isindependently substituted by lysine (K), arginine (R) or histidine (H).In one embodiment amino acid 124 is K, amino acid 147 is E, amino acid213 is E, and amino acid 123 is R. The aa substitutions are either inthe CD3 Fab or in one or two BCMA Fabs. Bispecific antibodies againstBCMA and CD3 as charge variants are described in EP14179705, disclosedby reference (further called as “charge variants resp. charge variantexchange”).

All amino acid numbering herein is according to Kabat (Kabat, E. A. etal, Sequences of Proteins of Immunological Interest, 5th ed. PublicHealth Service, National Institutes of Health, Bethesda, Md. (1991), NIHPublication 91-3242).

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of a singleamino acid composition.

The “antibodies” according to the invention can be of any class (e.g.IgA, IgD, IgE, IgG, and IgM, preferably IgG or IgE), or subclass (e.g.,IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2, preferably IgG1), whereby bothantibodies, from which the bivalent bispecific antibody according to theinvention is derived, have an Fc part of the same subclass(e.g. IgG1,IgG4 and the like, preferably IgG1), preferably of the same allotype(e.g. Caucasian).

A “Fc part of an antibody” is a term well known to the skilled artisanand defined on the basis of papain cleavage of antibodies. In oneembodiment of the invention the bispecific antibody contain as Fc part,in one embodiment a Fc part derived from human origin and preferably allother parts of the human constant regions. The Fc part of an antibody isdirectly involved in complement activation, C1q binding, C3 activationand Fc receptor binding. While the influence of an antibody on thecomplement system is dependent on certain conditions, binding to C1q iscaused by defined binding sites in the Fc part. Such binding sites areknown in the state of the art and described e.g. by Lukas, T J., et al.,J. Immunol. 127 (1981) 2555-2560; Brunhouse, R., and Cebra, J. J., Mol.Immunol. 16 (1979) 907-917; Burton, D. R., et al., Nature 288 (1980)338-344; Thommesen, J. E., et al., Mol. Immunol. 37 (2000) 995-1004;Idusogie, E. E., et al., J. Immunol. 164 (2000) 4178-4184; Hezareh, M.,et al., J. Virol. 75 (2001) 12161-12168; Morgan, A., et al., Immunology86 (1995) 319-324; and EP 0 307 434.

Such binding sites are e.g. L234, L235, D270, N297, E318, K320, K322,P331 and P329 (numbering according to EU index of Kabat). Antibodies ofsubclass IgG1, IgG2 and IgG3 usually show complement activation, C1 qbinding and C3 activation, whereas IgG4 do not activate the complementsystem, do not bind C1q and do not activate C3. In one embodiment the Fcpart is a human Fc part.

In one embodiment of the invention the bispecific antibody comprises anFc variant of a wild-type human IgG Fc region, said Fc variantcomprising an amino acid substitution at position Pro329 and at leastone further amino acid substitution, wherein the residues are numberedaccording to the EU index of Kabat, and wherein said antibody exhibits areduced affinity to the human FcγRIIIA and/or FcγRIIA and/or FcγRIcompared to an antibody comprising the wildtype IgG Fc region, andwherein the ADCC induced by said antibody is reduced to at least 20% ofthe ADCC induced by the antibody comprising a wild-type human IgG Fcregion. In a specific embodiment Pro329 of a wild-type human Fc regionin the bispecific antibody is substituted with glycine or arginine or anamino acid residue large enough to destroy the proline sandwich withinthe Fc/Fcγ receptor interface, that is formed between the proline329 ofthe Fc and tryptophane residues Trp 87 and Tip 110 of FcγRIII(Sondermann et al.: Nature 406, 267-273 (20 Jul. 2000)). In a furtheraspect of the invention the at least one further amino acid substitutionin the Fc variant is S228P, E233P, L234A, L235A, L235E, N297A, N297D, orP331S and still in another embodiment said at least one further aminoacid substitution is L234A and L235A of the human IgG1 Fc region orS228P and L235E of the human IgG4 Fc region. Such Fc variants aredescribed in detail in WO2012130831.

By “effector function” as used herein is meant a biochemical event thatresults from the interaction of an antibody Fc region with an Fcreceptor or ligand. Effector functions include but are not limited toADCC, ADCP, and CDC. By “effector cell” as used herein is meant a cellof the immune system that expresses one or more Fc receptors andmediates one or more effector functions. Effector cells include but arenot limited to monocytes, macrophages, neutrophils, dendritic cells,eosinophils, mast cells, platelets, B cells, large granular lymphocytes,Langerhans' cells, natural killer (NK) cells, and γδ T cells, and may befrom any organism including but not limited to humans, mice, rats,rabbits, and monkeys. By “library” herein is meant a set of Fc variantsin any form, including but not limited to a list of nucleic acid oramino acid sequences, a list of nucleic acid or amino acid substitutionsat variable positions, a physical library comprising nucleic acids thatencode the library sequences, or a physical library comprising the Fcvariant proteins, either in purified or unpurified form.

By “Fc gamma receptor” or “FcγR” as used herein is meant any member ofthe family of proteins that bind the IgG antibody Fc region and aresubstantially encoded by the FcγR genes. In humans this family includesbut is not limited to FcγRI (CD64), including isoforms FcγRIa, FcγRIb,and FcγRIc; FcγRII (CD32), including isoforms FcγRIIa (includingallotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2),and FcγRIIc; and FcγRIII (CD16), including isoforms FcγRIIIa (includingallotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1and FcγRIIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65), aswell as any undiscovered human FcγRs or FcγR isoforms or allotypes. AnFcγR may be from any organism, including but not limited to humans,mice, rats, rabbits, and monkeys. Mouse FcγRs include but are notlimited to FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2(CD16-2), as well as any undiscovered mouse FcγRs or FcγR isoforms orallotypes.

“Fc variant with increased effector function” as used herein is meant anFc sequence that differs from that of a parent Fc sequence by virtue ofat least one amino acid modification or relates to other modificationslike amendment of glycosylation at e.g. Asn279 that increase effectorfunctions. Such modifications are e.g. mentioned in Duncan et al., 1988,Nature 332:563-564; Lund et al., 1991, J Immunol 147:2657-2662; Lund etal., 1992, Mol Immunol 29:53-59; Alegre et al., 1994, Transplantation57:1537-1543; Hutchins et al., 1995, Proc Natl Acad Sci USA92:11980-11984; Jefferis et al., 1995, //77muno/Lett 44:111-117; Lund etal., 1995, Faseb J 9:115-119; Jefferis et al., 1996, Immunol Lett54:101-104; Lund et al., 1996, J Immunol 157:4963-4969; Armour et al.,1999, Eur J Immunol 29:2613-2624; Idusogie et al., 2000, J Immunol164:4178-4184; Reddy et al., 2000, J Immunol 164:1925-1933; Xu et al.,2000, Cell Immunol 200: 16-26; Idusogie et al., 2001, J Immunol166:2571-2575; Shields et al., 2001, J Biol Chem 276:6591-6604; Jefferiset al., 2002, Immunol Lett 82:57-65; Presta et al., 2002, Biochem SocTrans 30:487-490; U.S. Pat. Nos. 5,624,821; 5,885,573; 6,194,551;WO200042072; WO199958572. Such Fc modifications also include accordingto the invention engineered glycoforms of the Fc part. By “engineeredglycoform” as used herein is meant a carbohydrate composition that iscovalently attached to an Fc polypeptide, wherein said carbohydratecomposition differs chemically from that of a parent Fc polypeptide.Engineered glycoforms may be generated by any method, for example byusing engineered or variant expression strains, by co-expression withone or more enzymes, for example D1-4-N-acetylglucosaminyltransferaseIII (GnTIII), by expressing an Fc polypeptide in various organisms orcell lines from various organisms, or by modifying carbohydrate(s) afterthe Fc polypeptide has been expressed. Methods for generating engineeredglycoforms are known in the art and mentioned in Umana et al., 1999, NatBiotechnol 17:176-180; Davies et al., 2001, Biotechnol Bioeng74:288-294; Shields et al., 2002, J Biol Chem 277:26733-26740; Shinkawaet at, 2003, J Biol Chem 278:3466-3473) U.S. Pat. No. 6,602,684;WO200061739; WO200129246; WO200231140; WO200230954; Potelligent™technology (Biowa, Inc., Princeton, N.J.); GlycoMAb™ glycosylationengineering technology (GLYCART biotechnology AG, Zurich, Switzerland)).Engineered glycoform typically refers to the different carbohydrate oroligosaccharide composition than the parent Fc polypeptide. In oneembodiment of the invention the bispecific antibody comprises a Fcvariant with increased effector function show high binding affinity tothe Fc gamma receptor III (FcγRIII, CD 16a). High binding affinity toFcγRIII denotes that binding is enhanced for CD16a/F158 at least 10-foldin relation to the parent antibody (95% fucosylation) as referenceexpressed in CHO host cells, such as CHO DG44 or CHO K1 cells, or/andbinding is enhanced for CD16a/V158 at least 20-fold in relation to theparent antibody measured by Surface Plasmon Resonance (SPR) usingimmobilized CD 16a at an antibody concentration of 100 nM. FcγRIIIbinding can be increased by methods according to the state of the art,e.g. by modifying the amino acid sequence of the Fc part or theglycosylation of the Fc part of the antibody (see e.g. EP2235061). Mori,K et al., Cytotechnology 55 (2007)109 and Satoh M, et al., Expert OpinBiol Ther. 6 (2006) 1161-1173 relate to a FUT8(α-1,6-fucosyltransferase) gene knockout CHO line for the generation ofafucosylated antibodies.

The term “chimeric antibody” refers to an antibody comprising a variableregion, i.e., binding region, from one source or species and at least aportion of a constant region derived from a different source or species,usually prepared by recombinant DNA techniques. Chimeric antibodiescomprising a murine variable region and a human constant region arepreferred. Other preferred forms of “chimeric antibodies” encompassed bythe present invention are those in which the constant region has beenmodified or changed from that of the original antibody to generate theproperties according to the invention, especially in regard to C1qbinding and/or Fc receptor (FcR) binding. Such chimeric antibodies arealso referred to as “class-switched antibodies”. Chimeric antibodies arethe product of expressed immunoglobulin genes comprising DNA segmentsencoding immunoglobulin variable regions and DNA segments encodingimmunoglobulin constant regions. Methods for producing chimericantibodies involve conventional recombinant DNA and gene transfectiontechniques are well known in the art. See, e.g., Morrison, S. L., etal., Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855; U.S. Pat. Nos.5,202,238 and 5,204,244.

The term “humanized antibody” refers to antibodies in which theframework or “complementarity determining regions” (CDR) have beenmodified to comprise the CDR of an immunoglobulin of differentspecificity as compared to that of the parent immunoglobulin. In apreferred embodiment, a murine CDR is grafted into the framework regionof a human antibody to prepare the “humanized antibody.” See, e.g.,Riechmann, L., et al., Nature 332 (1988) 323-327; and Neuberger, M. S.,et al., Nature 314 (1985) 268-270. Other forms of “humanized antibodies”encompassed by the present invention are those in which the constantregion has been additionally modified or changed from that of theoriginal antibody to generate the properties according to the invention,especially in regard to Clq binding and/or Fc receptor (FcR) binding.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies are well-known in thestate of the art (van Dijk, M. A., and van de Winkel, J. G., Curr. Opin.Chem. Biol. 5 (2001) 368-374). Human antibodies can also be produced intransgenic animals (e.g., mice) that are capable, upon immunization, ofproducing a full repertoire or a selection of human antibodies in theabsence of endogenous immunoglobulin production. Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant mice willresult in the production of human antibodies (see, e.g., Jakobovits, A.,et al., Proc. Natl. Acad. Sci. USA 90 (1993) 2551-2555; Jakobovits, A.,et al., Nature 362 (1993) 255-258; Bruggemann, M., et al., Year Immunol.7 (1993) 33-40). Human antibodies can also be produced in phage displaylibraries (Hoogenboom, H. R., and Winter, G., J. Mol. Biol. 227 (1992)381-388; Marks, J. D., et al., J. Mol. Biol. 222 (1991) 581-597). Thetechniques of Cole et al. and Boerner et al. are also available for thepreparation of human monoclonal antibodies (Cole et al., MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); and Boerner,P., et al., J. Immunol. 147 (1991) 86-95). As already mentioned forchimeric and humanized antibodies according to the invention the term“human antibody” as used herein also comprises such antibodies which aremodified in the constant region to generate the properties according tothe invention, especially in regard to C1q binding and/or FcR binding,e.g. by “class switching” i.e. change or mutation of Fc parts (e.g. fromIgG1 to IgG4 and/or IgG1/IgG4 mutation.)

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies isolated from a hostcell such as a NSO or CHO cell or from an animal (e.g. a mouse) that istransgenic for human immunoglobulin genes or antibodies expressed usinga recombinant expression vector transfected into a host cell. Suchrecombinant human antibodies have variable and constant regions in arearranged form. The recombinant human antibodies have been subjected toin vivo somatic hypermutation. Thus, the amino acid sequences of the VHand VL regions of the recombinant antibodies are sequences that, whilederived from and related to human germ line VH and VL sequences, may notnaturally exist within the human antibody germ line repertoire in vivo.

The “variable domain” (variable domain of a light chain (VL), variableregion of a heavy chain (VH)) as used herein denotes each of the pair oflight and heavy chains which is involved directly in binding theantibody. The domains of variable human light and heavy chains have thesame general structure and each domain comprises four framework (FR)regions whose sequences are widely conserved, connected by three“hypervariable regions” (or complementarity determining regions, CDRs).The framework regions adopt a β-sheet conformation and the CDRs may formloops connecting the β-sheet structure. The CDRs in each chain are heldin their three-dimensional structure by the framework regions and formtogether with the CDRs from the other chain the binding site. Theantibody heavy and light chain CDR3 regions play a particularlyimportant role in the binding specificity/affinity of the antibodies andtherefore provide a further object of the invention. “A binding partcomprising a VH region and a VL region” means that these regions are therespective VH and VL regions of said BCMA or CD3 binding part.

The terms “hypervariable region” or “target-binding portion of anantibody” when used herein refer to the amino acid residues of anantibody which are responsible for target-binding. The hypervariableregion comprises amino acid residues from the “complementaritydetermining regions” or “CDRs”. “Framework” or “FR” regions are thosevariable domain regions other than the hypervariable region residues asherein defined. Therefore, the light and heavy chains of an antibodycomprise from N- to C-terminus the domains FR1, CDR1, FR2, CDR2, FR3,CDR3, and FR4. CDRs on each chain are separated by such framework aminoacids. Especially, CDR3 of the heavy chain is the region whichcontributes most to target binding. CDR and FR regions are determinedaccording to the standard definition of Kabat et al., Sequences ofProteins of Immunological Interest, 5th ed., Public Health Service,National Institutes of Health, Bethesda, Md. (1991). The terms “CDR1H,CDR2H and CDR3H” as used herein refer to the respective CDRs of theheavy chain located in the variable domain VH. The terms “CDR1L, CDR2Land CDR3L” as used herein refer to the respective CDRs of the lightchain located in the variable domain VL.

The constant heavy chain domain CH1 by which the heavy chain domain CH3is replaced can be of any Ig class (e.g. IgA, IgD, IgE, IgG, and IgM),or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2). The constantlight chain domain CL by which the heavy chain domain CH3 is replacedcan be of the lambda (λ) or kappa (κ) type, preferably the kappa (κ)type.

The term “target” or “target molecule” as used herein are usedinterchangeable and refer to human BCMA. In regard to bispecificantibodies the term refers to BCMA and the second target. Preferably inregard to bispecific antibodies the term refers to BCMA and CD3.

The term “epitope” includes any polypeptide determinant capable ofspecific binding to an antibody. In certain embodiments, epitopedeterminant include chemically active surface groupings of moleculessuch as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, incertain embodiments, may have specific three dimensional structuralcharacteristics, and or specific charge characteristics. An epitope is aregion of a target that is bound by an antibody.

In general there are two vectors encoding the light chain and heavychain of an antibody. In regard to a bispecific antibody there are twovectors encoding the light chain and heavy chain of said antibodyspecifically binding to the first target, and further two vectorsencoding the light chain and heavy chain of said antibody specificallybinding to the second target. One of the two vectors is encoding therespective light chain and the other of the two vectors is encoding therespective heavy chain. However in an alternative method for thepreparation of an antibody, only one first vector encoding the lightchain and heavy chain of the antibody specifically binding to the firsttarget and only one second vector encoding the light chain and heavychain of the antibody specifically binding to the second target can beused for transforming the host cell.

The term “nucleic acid or nucleic acid molecule”, as used herein, isintended to include DNA molecules and RNA molecules. A nucleic acidmolecule may be single-stranded or double-stranded, but preferably isdouble-stranded DNA.

As used herein, the expressions “cell,” “cell line,” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations. Variant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded. Where distinct designations are intended, it will be clearfrom the context.

The term “transformation” as used herein refers to process of transferof a vectors/nucleic acid into a host cell. If cells without formidablecell wall barriers are used as host cells, transfection is carried oute.g. by the calcium phosphate precipitation method as described byGraham and Van der Eh, Virology 52 (1978) 546ff. However, other methodsfor introducing DNA into cells such as by nuclear injection or byprotoplast fusion may also be used. If prokaryotic cells or cells whichcontain substantial cell wall constructions are used, e.g. one method oftransfection is calcium treatment using calcium chloride as described byCohen S N, et al, PNAS 1972, 69 (8): 2110-2114.

Recombinant production of antibodies using transformation is well-knownin the state of the art and described, for example, in the reviewarticles of Makrides, S. C, Protein Expr. Purif. 17 (1999) 183-202;Geisse, S., et al., Protein Expr. Purif. 8 (1996) 271-282; Kaufman, RJ., Mol. Biotechnol. 16 (2000) 151-161; Werner, R. G., et al.,Arzneimittelforschung 48 (1998) 870-880 as well as in U.S. Pat. Nos.6,331,415 and 4,816,567.

As used herein, “expression” refers to the process by which a nucleicacid is transcribed into mRNA and/or to the process by which thetranscribed mRNA (also referred to as transcript) is subsequently beingtranslated into peptides, polypeptides, or proteins. The transcripts andthe encoded polypeptides are collectively referred to as gene product.If the polynucleotide is derived from genomic DNA, expression in aeukaryotic cell may include splicing of the mRNA.

A “vector” is a nucleic acid molecule, in particular self-replicating,which transfers an inserted nucleic acid molecule into and/or betweenhost cells. The term includes vectors that function primarily forinsertion of DNA or RNA into a cell (e.g., chromosomal integration),replication of vectors that function primarily for the replication ofDNA or RNA, and expression vectors that function for transcriptionand/or translation of the DNA or RNA. Also included are vectors thatprovide more than one of the functions as described.

An “expression vector” is a polynucleotide which, when introduced intoan appropriate host cell, can be transcribed and translated into apolypeptide. An “expression system” usually refers to a suitable hostcell comprised of an expression vector that can function to yield adesired expression product.

The antibodies are preferably produced by recombinant means. Suchmethods are widely known in the state of the art and comprise proteinexpression in prokaryotic and eukaryotic cells with subsequent isolationof the antibody polypeptide and usually purification to apharmaceutically acceptable purity. For the protein expression, nucleicacids encoding light and heavy chains or fragments thereof are insertedinto expression vectors by standard methods. Expression is performed inappropriate prokaryotic or eukaryotic host cells like CHO cells, NSOcells, SP2/0 cells, HEK293 cells, COS cells, yeast, or E. coli cells,and the antibody is recovered from the cells (supernatant or cells afterlysis). The bispecific antibodies may be present in whole cells, in acell lysate, or in a partially purified or substantially pure form.Purification is performed in order to eliminate other cellularcomponents or other contaminants, e.g. other cellular nucleic acids orproteins, by standard techniques, including alkaline/SDS treatment,column chromatography and others well known in the art. See Ausubel, F.,et al., ed., Current Protocols in Molecular Biology, Greene Publishingand Wiley Interscience, New York (1987).

Expression in NS0 cells is described by, e.g., Barnes, L. M., et al.,Cytotechnology 32 (2000) 109-123; and Barnes, L. M., et al., Biotech.Bioeng. 73 (2001) 261-270. Transient expression is described by, e.g.,Durocher, Y., et al., Nucl. Acids. Res. 30 (2002) E9. Cloning ofvariable domains is described by Orlandi, R., et al., Proc. Natl. Acad.Sci. USA 86 (1989) 3833-3837; Carter, P., et al., Proc. Natl. Acad. Sci.USA 89 (1992) 4285-4289; and Norderhaug, L., et al., J. Immunol. Methods204 (1997) 77-87. A preferred transient expression system (HEK293) isdescribed by Schlaeger, E.- J., and Christensen, K., in Cytotechnology30 (1999) 71-83 and by Schlaeger, E.-J., in J. Immunol. Methods 194(1996) 191-199.

The control sequences that are suitable for prokaryotes, for example,include a promoter, optionally an operator sequence, and a ribosomebinding site. Eukaryotic cells are known to utilize promoters, enhancersand polyadenylation signals.

The antibodies are suitably separated from the culture medium byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography. DNA or RNAencoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures. The hybridoma cells can serve as a sourceof such DNA and RNA. Once isolated, the DNA may be inserted intoexpression vectors, which are then transfected into host cells such asHEK293 cells, CHO cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of recombinantmonoclonal antibodies in the host cells.

Amino acid sequence variants (or mutants) of an antibody are prepared byintroducing appropriate nucleotide changes into the antibody DNA, or bynucleotide synthesis. Such modifications can be performed, however, onlyin a very limited range, e.g. as described above. For example, themodifications do not alter the above mentioned antibody characteristicssuch as the IgG isotype and target binding, but may improve the yield ofthe recombinant production, protein stability or facilitate thepurification.

In one embodiment of the invention, instead of the bispecific antibody,there is used a chimeric antigen receptor (CAR), wherein the CARcomprises an antigen recognition moiety directed against BCMA, atransmembrane moiety and a T-cell activation moiety, characterized inthat the antigen recognition moiety is an antibody according to theinvention (here not the bispecific antibody). Such CAR is then usuallytransferred by using a vector, preferably a retroviral vector,comprising the sequence encoding said CAR, into an immune effector cellfor which herein the term “a CAR T-cell” as used. Such CAR T-cells arethen used in combination with said immunotherapeutic drug.

The encoded antibody can be also an antigen binding fragment thereof asspecified. Structures and generation of such “BCMA CARs” are describede.g. in WO2013154760, WO2015052538, WO2015090229, and WO2015092024.

In one embodiment, instead of the bispecific antibody, there is used achimeric antigen receptor (CAR) or the respective CAR T-cell,comprising:

(i) a B cell maturation antigen (BCMA) recognition moiety;

(ii) a spacer domain; and

(ii) a transmembrane domain; and

(iii) an intracellular T cell signaling domain,

characterized in that the BCMA recognition moiety is a monoclonalantibody specifically binding to BCMA, characterized in comprising aCDR3H region of SEQ ID NO:17 and a CDR3L region of SEQ ID NO:20 and aCDR1H, CDR2H, CDR1L, and CDR2L region combination selected from thegroup of

a) CDR1H region of SEQ ID NO:21 and CDR2H region of SEQ ID NO:22, CDR1Lregion of SEQ ID NO:23, and CDR2L region of SEQ ID NO:24,

b) CDR1H region of SEQ ID NO:21 and CDR2H region of SEQ ID NO:22, CDR1Lregion of SEQ ID NO:25, and CDR2L region of SEQ ID NO:26,

c) CDR1H region of SEQ ID NO:21 and CDR2H region of SEQ ID NO:22, CDR1Lregion of SEQ ID NO:27, and CDR2L region of SEQ ID NO:28,

d) CDR1H region of SEQ ID NO:29 and CDR2H region of SEQ ID NO:30, CDR1Lregion of SEQ ID NO:31, and CDR2L region of SEQ ID NO:32,

e) CDR1H region of SEQ ID NO:34 and CDR2H region of SEQ ID NO:35, CDR1Lregion of SEQ ID NO:31, and CDR2L region of SEQ ID NO:32, and

f) CDR1H region of SEQ ID NO:36 and CDR2H region of SEQ ID NO:37, CDR1Lregion of SEQ ID NO:31, and CDR2L region of SEQ ID NO:32.

The T-cell activation moiety can be any suitable moiety derived orobtained from any suitable molecule. In one embodiment, for example, theT-cell activation moiety comprises a transmembrane domain. Thetransmembrane domain can be any transmembrane domain derived or obtainedfrom any molecule known in the art. For example, the transmembranedomain can be obtained or derived from a CD8a molecule or a CD28molecule. CD8 is a transmembrane glycoprotein that serves as aco-receptor for the T-cell receptor (TCR), and is expressed primarily onthe surface of cytotoxic T-cells. The most common form of CD8 exists asa dimer composed of a CD8 alpha and CD8 beta chain. CD28 is expressed onT-cells and provides co-stimulatory signals required for T-cellactivation. CD28 is the receptor for CD80 (B7.1) and CD86 (B7.2). In apreferred embodiment, the CD8 alpha and CD28 are human. In addition tothe transmembrane domain, the T-cell activation moiety further comprisesan intracellular (i.e., cytoplasmic) T-cell signaling domain. Theintercellular T-cell signaling domain can be obtained or derived from aCD28 molecule, a CD3 zeta molecule or modified versions thereof, a humanFc receptor gamma (FcRy) chain, a CD27 molecule, an OX40 molecule, a4-IBB molecule, or other intracellular signaling molecules known in theart. As discussed above, CD28 is a T-cell marker important in T-cellco-stimulation. CD3 zeta, associates with TCRs to produce a signal andcontains immunoreceptor tyrosine-based activation motifs (ITAMs). 4-1BB,also known as CD137, transmits a potent costimulatory signal to T-cells,promoting differentiation and enhancing long-term survival of Tlymphocytes. In one embodiment, the CD28, CD3 zeta, 4-1BB, OX40, andCD27 are human

Such CAR or the respective CAR T-cell is together with theimmunotherapeutic drug, described herein, for use

a) in the combined use in treating multiple myeloma according to theinvention,

b) in the method of treating multiple myeloma according to theinvention,

c) in the therapeutic combination for achieving multiple myeloma celllysis in a patient suffering from multiple myeloma disease according tothe invention,

d) in the article of manufacture according to the invention, and

e) in the method for manufacturing a medicament according to theinvention.

T cell bispecific (TCB) binders have very highconcentration/tumor-cell-receptor-occupancy dependent potency in cellkilling (e.g. EC₅₀ in in vitro cell killing assays in the sub- or lowpicomolar range; Dreier et al. Int J Cancer 2002), T-cell bispecificbinder (TCB) are given at much lower doses than conventionalmonospecific antibodies. For example, blinatumomab (CD19×CD3) is givenat a continuous intravenous dose of 5 to 15 μg/m²/day (i.e. only 0.35 to0.105 mg/m²/week) for treatment of acute lymphocytic leukemia or 60μg/m²/day for treatment of Non Hodgkin Lymphoma, and the serumconcentrations at these doses are in the range of 0.5 to 4 ng/ml(Klinger et al., Blood 2012; Topp et al., J Clin Oncol 2011; Goebeler etal. Ann Oncol 2011). Because low doses of TCB can exert high efficacy inpatients, it is envisaged that for the bispecific antibody and the drugsubcutaneous administration is possible and preferred in the clinicalsettings (preferably in the dose range of 0.1 to 2.5, preferably 25mg/m²/week, preferably 250 mg/m2/week). Even at these lowconcentrations/doses/receptor occupancies, TCB can cause considerableadverse events (Klinger et al., Blood 2012). Therefore it is critical tocontrol tumor cell occupancy/coverage. In patients with high andvariable levels of serum APRIL and BAFF (e.g. multiple myeloma patients,Moreaux et al. 2004; Blood 103(8): 3148-3157) number of TCB bound to thetumor cells resp. tumor cell occupancy may be considerably influenced byAPRIL/BAFF. But by using said antibody used in this invention, tumorcell occupancy respectively efficacy/safety it may not be required toincrease the dose for an antibody according to this invention as saidantibody may not be affected by APRIL/BAFF ligand competition. Anotheradvantage of the bispecific antibody is based on the inclusion of an Fcportion, which increases the elimination half-life to about 4 to 12 daysand allows at least once or twice/week administrations as compared toTCBs without an Fc portion (e.g. blinatumomab) which are required to begiven intravenously and continuously with a pump carried by patients.

The biological properties of the bispecific antibodies have beeninvestigated in several studies in comparison to 83A10-TCBcv. Thepotency to induce T-cell redirected cytotoxicity of e.g.anti-BCMA/anti-CD3 TCB antibodies 21-TCBcv, 22-TCBcv, 42-TCBcv incomparison to 83A10-TCBcv was measured on H929 MM cell line (Example 8,Table 12, FIG. 4). The antibodies used in this invention were studiedand analysis showed that concentration dependent killing of H929 cellsresp. the EC50 values were found to be higher than EC50 valuesdetermined for 83A10-TCBcv; suggesting that the bispecific antibodieswere less potent to induce killing of H929 MM cells than Mab 83A10 asTCB. Surprisingly a turnover was observed when T-cell redirectedcytotoxicity was measured on RPMI-8226 MM cell line and also JJN-3 cellline (respectively, examples 10 and 11, Tables 13, and 14 and 15, FIGS.6 and 7): the bispecific antibodies showed lower EC50 and thereforehigher potency than 83A10-TCBcv. To the surprise of the inventors, thebispecific antibodies showed several advantages in a direct comparisonwith 83A10 TCBcv in bone marrow aspirates freshly taken from MM patients(note: to get the best possible comparison, in all bone marrow aspiratesalways all T-cell bispecific (TCB) antibodies have been tested at sameconcentrations);

-   -   Higher killing potency of myeloma cells, i.e. same % of killing        already at lower concentrations than with 83A10-TCBcv        respectively concentration response curves for killing shifted        to the left (Example 13, Tables 18, 19 and 20, FIGS. 8, 9 and        10). Already at a concentration of 1 nM of the bispecific        antibodies in seven different patient bone marrow aspirates        reduction relative to control of propidium iodide negative        viable multiple myeloma cancer cells was between 77.1 and 100%.        With 1 nM 83A10-TCBcv in same seven bone marrow aspirates        reductions of only 37.1 to 98.3% have been achieved (Tables 20        and 21).    -   Higher maximal killing as compared to 83A10-TCBcv was achieved        at the highest concentration tested (10 nM) in the same        experiment with the seven (7) bone marrow aspirates for the        bispecific antibodies (Tables 20 and 21).    -   Non responders to 83A10-TCBcv can be turned to responders if        22-TCBcv/42-TCBcv are used: In two (2) bone marrow patient        samples in which no killing response to 83A10-TCBcv was        observed, surprisingly killing could be found with antibodies as        TCBs according to the invention (FIGS. 9A and 9B).

The BCMA×CD3 TCB used in this invention bind to human and cynomolgusmonkeys (cyno) BCMA and to BCMA of mice and rat, appropriate fortoxicological examination in cynomolgus monkeys if the CD3 binder alsobinds to cynomolgus CD3 or in mouse/rat if the CD3 binder also binds tomouse/rat BCMA. Surprisingly the binding affinity to cyno BCMA is veryclose to the binding affinity to human BCMA. SPR has been used tomeasure binding affinities to human and cyno BCMA (Example 2, Table 4).Cyno/human gap (ratio of affinity for cyno to human BCMA, KD) has beencalculated from measured affinity data by dividing affinity to cyno BCMAthrough affinity to human BCMA (Example 3, Table 5). For 83A10 acyno/human gap of 15.3 was found (i.e. 15.3 times lower binding affinityto cyno than to human BCMA). To the surprise of the inventors thebispecific antibodies showed cyno/human gaps between 15.4 and 1.7, whichis similar or in majority more favorable cyno/human gap than that of83A10 (Table 5). Because the CD3 binder used in the BCMA×CD3 TCB iscross-reactive to cynomolgus monkey CD3, pharmacokinetics andpharmacodynamics investigations can be obtained from cynomolgus monkeys(see Example 16). Also toxicological investigations in cynomolgusmonkeys are predictive of the pharmacological and toxicological effectsin humans and the cross-reactivity to cynomolgus monkeys feature is tothe benefit of patients. The BCMA antibodies used in this invention alsobind to murine BCMA (e.g. Kd of clones 22 and 42 measured by SPR as 0.9nM and 2.5 nM) see table 2D in Example 1.1.1A.4). The CD3 binder of theBCMA×CD3 TCB is not cross-reactive to murine CD3.

In summary the potency and efficacy advantages for killing of low BCMAexpressing MM cell lines like RPMI-8226 and JJN-3 and especially forkilling of MM cells in patient bone marrow aspirates and in addition thevery favorable cyno/human gap in binding affinities to BCMA make theantibodies used in this invention and respective TCBs essentiallypromising agents for treatment of MM patients. In addition theanti-BCMA×CD3 TCBcv used in this invention have, as 83A10-TCBcv,favorable properties like long elimination half-life, efficacy at once aweek administration (intravenously, subcutaneously), low or no tendencyto aggregation and can be manufactured with high purity and good yield.

TABLE 1A Antibody sequences SEQ ID NO: Name(s) aa sequences  1 CD3 CDR1HTYAMN  2 CD3 CDR2H RIRSKYNNYATYYADSVKG  3 CD3 CDR3H HGNFGNSYVSWFAY  4CD3 CDR1L GSSTGAVTTSNYAN  5 CD3 CDR2L GTNKRAP  6 CD3 CDR3L ALWYSNLWV  7CD3 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFA YWGQGTLVTVSS  8 CD3 VLQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALT LSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVL 9 83A10 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVLGWFDYWGQGTLV TVSS 10 Mab21 VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSDNAMGWV Mab22 VHRQAPGKGLEWVSAISGPGSSTYYADSVKGRFTISRDNSK Mab42 VHNTLYLQMNSLRAEDTAVYYCAKVLGWFDYWGQGTLV TVSS 11 83A10 VLEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLE PEDFAVYYCQQYGYPPDFTFGQGTKVEIK12 Mab21 VL EIVLTQSPGTLSLSPGERATLSCRASQSVSEYYLAWYQQ Mab27 VLKPGQAPRLLIEHASTRATGIPDRFSGSGSGTDFTLTISRLE Mab33 VLPEDFAVYYCQQYGYPPDFTFGQGTKVEIK Mab39 VL 13 Mab22 VLEIVLTQSPGTLSLSPGERATLSCRASQSVSSYYLAWYQQKPGQAPRLLISGAGSRATGIPDRFSGSGSGTDFTLTISRLE PEDFAVYYCQQYGYPPDFTFGQGTKVEIK14 Mab42 VL EIVLTQSPGTLSLSPGERATLSCRASQSVSDEYLSWYQQKPGQAPRLLIHSASTRATGIPDRFSGSGSGTDFTLAISRLE PEDFAVYYCQQYGYPPDFTFGQGTKVEIK15 83A10 CDR1H SYAMS 16 83A10 CDR2H AISGSGGSTYYADSVKG 17 83A10 CDR3HVLGWFDY Mab21 CDR3H Mab22 CDR3H Mab42 CDR3H Mab27 CDR3H Mab33 CDR3HMab39 CDR3H 18 83A10 CDR1L RASQSVSSSYLAW 19 83A10 CDR2L YGASSRAT 2083A10 CDR3L QQYGYPPDFT Mab21 CDR3L Mab22 CDR3L Mab42 CDR3L 21Mab21 CDR1H DNAMG Mab22 CDR1H Mab42 CDR1H 22 Mab21 CDR2HAISGPGSSTYYADSVKG Mab22 CDR2H Mab42 CDR2H 23 Mab21 CDR1L RASQSVSEYYLAW24 Mab21 CDR2L EHASTRAT 25 Mab22 CDR1L RASQSVSSYYLAW 26 Mab22 CDR2LSGAGSRAT 27 Mab42 CDR1L RASQSVSDEYLSW 28 Mab42 CDR2L HSASTRAT 29Mab27 CDR1H SAPMG 30 Mab27 CDR2H AISYIGHTYYADSVKG 31 Mab27 CDR1LRASQSVSEYYLA Mab33 CDR1L Mab39 CDR1L 32 Mab27 CDR2L HASTRAT Mab33 CDR2LMab39 CDR2L 33 Mab27 CDR3L QQYGYPPDFT Mab33 CDR3L Mab39 CDR3L 34Mab33 CDR1H TNAMG 35 Mab33 CDR2H AINRFGGSTYYADSVKG 36 Mab39 CDR1H QNAMG37 Mab39 CDR2H AISPTGFSTYYADSVKG 38 Mab27 VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSSAPMGWVRQAPGKGLEWVSAISYIGHTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVLGWFDYWGQGTLVTV SS 39 Mab33 VHEVQLLESGGGLVQPGGSLRLSCAASGFTFYTNAMGWVRQAPGKGLEWVSAINRFGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVLGWFDYWGQGTL VTVSS 40 Mab39 VHEVQLLESGGGLVQPGGSLRLSCAASGFTFTQNAMGWVRQAPGKGLEWVSAISPTGFSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVLGWFDYWGQGTLV TVSS 41 83A10 BCMA CH1ASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTV Mab21 BCMA CH1SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT Mab22 BCMA CH1QTYICNVNHKPSNTKVDEKVEPKSC Mab42 BCMA CH1 42 83A10 BCMA CLRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKV Mab21 BCMA CLQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA Mab22 BCMA CLDYEKHKVYACEVTHQGLSSPVTKSFNRGEC Mab42 BCMA CL 43 CD3 CH1ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSC 44CD3 CL ASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 4583A10 knob HC EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVLGWFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK 4683A10 hole HC EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVLGWFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK 4783A10 LC EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGYPPDFTFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC 48 CD3 LCEVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC 49 Mab21 knob HCEVQLLESGGGLVQPGGSLRLSCAASGFTFSDNAMGWVRQAPGKGLEWVSAISGPGSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVLGWFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCGSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK 50Mab21 hole HC EVQLLESGGGLVQPGGSLRLSCAASGFTFSDNAMGWVRQAPGKGLEWVSAISGPGSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVLGWFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK 51Mab21 LC EIVLTQSPGTLSLSPGERATLSCRASQSVSEYYLAWYQQKPGQAPRLLIEHASTRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGYPPDFTFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC 52Mab22 knob HC EVQLLESGGGLVQPGGSLRLSCAASGFTFSDNAMGWVRQAPGKGLEWVSAISGPGSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVLGWFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK 53Mab22 hole HC EVQLLESGGGLVQPGGSLRLSCAASGFTFSDNAMGWVRQAPGKGLEWVSAISGPGSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVLGWFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK 54Mab22 LC EIVLTQSPGTLSLSPGERATLSCRASQSVSSYYLAWYQQKPGQAPRLLISGAGSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGYPPDFTFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC 55Mab42 knob HC EVQLLESGGGLVQPGGSLRLSCAASGFTFSDNAMGWVRQAPGKGLEWVSAISGPGSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVLGWFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK 56Mab42 hole HC EVQLLESGGGLVQPGGSLRLSCAASGFTFSDNAMGWVRQAPGKGLEWVSAISGPGSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVLGWFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK 57Mab42 LC EIVLTQSPGTLSLSPGERATLSCRASQSVSDEYLSWYQQKPGQAPRLLIHSASTRATGIPDRFSGSGSGTDFTLAISRLEPEDFAVYYCQQYGYPPDFTFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC

Remark: SEQ ID NO:20 and SEQ ID NO:33 are identical

TABLE 1B Antibody sequences (short list) SEQ ID NO: VH VL CDR1H CDR2HCDR3H CDR1L CDR2L CDR3L CD3 antibody 7 8 1 2 3 4 5 6 BCMA antibody 83A109 11 15 16 17 18 19 20 Mab21 10 12 21 22 17 23 24 20 Mab22 10 13 21 2217 25 26 20 Mab42 10 14 21 22 17 27 28 20 Mab27 38 12 29 30 17 31 32 33Mab33 39 12 34 35 17 31 32 33 Mab39 40 12 36 37 17 31 32 33

TABLE 2A Additional constructs SEQ ID NO: Fragment/Construct 83A10 Mab21Mab22 Mab42 BCMA CH1 41 41 41 41 BCMA CL 42 42 42 42 CD3 CH1 43 43 43 43CD3 CL 44 44 44 44

TABLE 2B Additional constructs SEQ ID NO: Construct 83A10 Mab21 Mab22Mab42 BCMA VH_CH1cv × CD3 45 49 52 55 VL_CH1 Fc knob LALA PG (knob HC)BCMAcv HC hole LALA PG 46 50 53 56 (hole HC) BCMAcv hum IgG1 LC (BCMA 4751 54 57 LC) CD3 VH_CL (CD3 LC) 48 48 48 48

To make the following (2+1) Fc-containing anti-BCMA/anti-CD3 TCBs, therespective constructs/sequence IDs as mentioned in the table 2B abovewere used:

83A10-TCBcv: 45, 46, 47 (×2), 48 (FIG. 2A)

21-TCBcv: 48, 49, 50, 51 (×2) (FIG. 2A)

22-TCBcv: 48, 52, 53, 54 (×2) (FIG. 2A)

42-TCBcv: 48, 55, 56, 57 (×2) (FIG. 2A)

In the following specific embodiments of the invention are listed:

I. A bispecific antibody comprising a first binding part specificallybinding to human B cell maturation antigen (BCMA) and a second bindingpart specifically binding to human CD3ε (CD3) and an immunotherapeuticdrug selected from the group consisting of thalidomide and animmunotherapeutic derivative thereof, an anti-CD38 antibody, ananti-PD-1 antibody and an anti-PD-L1 antibody, for combined use intreating multiple myeloma, characterized in that said first binding partcomprises a VH region comprising a CDR1H region of SEQ ID NO:21, a CDR2Hregion of SEQ ID NO:22 and a CDR3H region of SEQ ID NO:17 and a VLregion comprising a CDR3L region of SEQ ID NO:20 and a CDR1L and CDR2Lregion combination selected from the group of

-   -   i) CDR1L region of SEQ ID NO:23 and CDR2L region of SEQ ID        NO:24,    -   ii) CDR1L region of SEQ ID NO:25 and CDR2L region of SEQ ID        NO:26, or    -   iii) CDR1L region of SEQ ID NO:27 and CDR2L region of SEQ ID        NO:28.

II. The bispecific antibody and the immunotherapeutic drug for combineduse in treating multiple myeloma according to embodiment I,characterized in that the immunotherapeutic drug is selected from thegroup consisting of daratumumab, isatuximab, MOR202, Ab79, Ab19,thalidomide, lenalidomide, pomalidomide, pembrolizumab, pidilizumab,nivolumab, MEDI-0680, PDR001, REGN2810, lambrolizumab, MDX-1106,BGB-108, h409A11, h409A16, h409A17, and atezolizumab.

III. A method of treating multiple myeloma, characterized inadministering to a patient in need of such treatment

-   -   a) a bispecific antibody comprising a first binding part        specifically binding to human B cell maturation antigen (BCMA)        and a second binding part specifically binding to human CD3ε        (CD3) and    -   b) an immunotherapeutic drug selected form the group consisting        of thalidomide and an immunotherapeutic derivative thereof, an        anti-CD38 antibody, an anti-PD-1 antibody and an anti-PD-L1        antibody,        characterized in that said first binding part comprises a VH        region comprising a CDR1H region of SEQ ID NO:21, a CDR2H region        of SEQ ID NO:22 and a CDR3H region of SEQ ID NO:17 and a VL        region comprising a CDR3L region of SEQ ID NO:20 and a CDR1L and        CDR2L region combination selected from the group of    -   i) CDR1L region of SEQ ID NO:23 and CDR2L region of SEQ ID        NO:24,    -   ii) CDR1L region of SEQ ID NO:25 and CDR2L region of SEQ ID        NO:26, or    -   iii) CDR1L region of SEQ ID NO:27 and CDR2L region of SEQ ID        NO:28.

The bispecific antibody and the immunotherapeutic drug are used in atherapeutically effective amount.

IV. The method according to embodiment III, characterized in that theimmunotherapeutic drug is selected from the group consisting ofdaratumumab, isatuximab, MOR202, Ab79, Ab19, thalidomide, lenalidomide,pomalidomide, pembrolizumab, pidilizumab, nivolumab, MEDI-0680, PDR001,REGN2810, lambrolizumab, MDX-1106, BGB-108, h409A11, h409A16, h409A17,and atezolizumab.

V. A therapeutic combination for achieving multiple myeloma cell lysisin a patient suffering from multiple myeloma disease, characterized incomprising

a) a bispecific antibody comprising a first binding part specificallybinding to human B cell maturation antigen (BCMA) and a second bindingpart specifically binding to human CD3ε (CD3) and

b) an immunotherapeutic drug selected form the group consisting ofthalidomide and an immunotherapeutic derivative thereof, an anti-CD38antibody, an anti-PD-1 antibody and an anti-PD-L1 antibody,characterized in that said first binding part comprises a VH regioncomprising a CDR1H region of SEQ ID NO:21, a CDR2H region of SEQ IDNO:22 and a CDR3H region of SEQ ID NO:17 and a VL region comprising aCDR3L region of SEQ ID NO:20 and a CDR1L and CDR2L region combinationselected from the group of

i) CDR1L region of SEQ ID NO:23 and CDR2L region of SEQ ID NO:24,

ii) CDR1L region of SEQ ID NO:25 and CDR2L region of SEQ ID NO:26, or

iii) CDR1L region of SEQ ID NO:27 and CDR2L region of SEQ ID NO:28.

The bispecific antibody and the immunotherapeutic drug are used in atherapeutically effective amount.

VI. The therapeutic combination according to embodiment V, characterizedin that the immunotherapeutic drug is selected from the group consistingof daratumumab, isatuximab, MOR202, Ab79, Ab19, thalidomide,lenalidomide, pomalidomide, CC-122, CC-220, pembrolizumab, pidilizumab,nivolumab, MEDI-0680, PDR001, REGN2810, lambrolizumab, MDX-1106,BGB-108, h409A11, h409A16, h409A17, atezolizumab, avelumab, durvalumab,and MDX-1105.

VII. An article of manufacture, characterized in comprising

a) a bispecific antibody comprising a first binding part specificallybinding to human B cell maturation antigen (BCMA) and a second bindingpart specifically binding to human CD3ε (CD3), characterized in thatsaid first binding part comprises a VH region comprising a CDR1H regionof SEQ ID NO:21, a CDR2H region of SEQ ID NO:22 and a CDR3H region ofSEQ ID NO:17 and a VL region comprising a CDR3L region of SEQ ID NO:20and a CDR1L and CDR2L region combination selected from the group of

i) CDR1L region of SEQ ID NO:23 and CDR2L region of SEQ ID NO:24,

ii) CDR1L region of SEQ ID NO:25 and CDR2L region of SEQ ID NO:26, or

iii) CDR1L region of SEQ ID NO:27 and CDR2L region of SEQ ID NO:28. in apharmaceutically acceptable carrier,

b) an immunotherapeutic drug selected form the group consisting ofthalidomide and an immunotherapeutic derivative thereof, an anti-CD38antibody, an anti-PD-1 antibody and an anti-PD-L1 antibody,

c) a pharmaceutically acceptable carrier and instructions foradministering said bispecific antibody and said immunotherapeutic drugin combination to a subject in need of a treatment for multiple myeloma.

VIII. The article of manufacture according to embodiment VII,characterized in that the immunotherapeutic drug is selected from thegroup consisting of daratumumab, isatuximab, MOR202, Ab79, Ab19,thalidomide, lenalidomide, pomalidomide, CC-122, CC-220, pembrolizumab,pidilizumab, nivolumab, MEDI-0680, PDR001, REGN2810, lambrolizumab,MDX-1106, BGB-108, h409A11, h409A16, h409A17, atezolizumab, avelumab,durvalumab, and MDX-1105.

IX. A method for manufacturing a medicament, characterized in using

a)) a bispecific antibody comprising a first binding part specificallybinding to human B cell maturation antigen (BCMA) and a second bindingpart specifically binding to human CD3ε (CD3), characterized in thatsaid first binding part comprises a VH region comprising a CDR1H regionof SEQ ID NO:21, a CDR2H region of SEQ ID NO:22 and a CDR3H region ofSEQ ID NO:17 and a VL region comprising a CDR3L region of SEQ ID NO:20and a CDR1L and CDR2L region combination selected from the group of

i) CDR1L region of SEQ ID NO:23 and CDR2L region of SEQ ID NO:24,

ii) CDR1L region of SEQ ID NO:25 and CDR2L region of SEQ ID NO:26, or

iii) CDR1L region of SEQ ID NO:27 and CDR2L region of SEQ ID NO:28,

b) an immunotherapeutic drug selected form the group consisting ofthalidomide and an immunotherapeutic derivative thereof, an anti-CD38antibody, an anti-PD-1 antibody and an anti-PD-L1 antibody,

c) combining said bispecific antibody and said immunotherapeutic drug ina pharmaceutically acceptable carrier.

The bispecific antibody and the immunotherapeutic drug are used in atherapeutically effective amount.

X. The method for manufacturing a medicament according to embodiment IX,characterized in that the immunotherapeutic drug is selected from thegroup consisting of daratumumab, isatuximab, MOR202, Ab79, Ab19,thalidomide, lenalidomide, pomalidomide, CC-122, CC-220, pembrolizumab,pidilizumab, nivolumab, MEDI-0680, PDR001, REGN2810, lambrolizumab,MDX-1106, BGB-108, h409A11, h409A16, h409A17, atezolizumab, avelumab,durvalumab, and MDX-1105.

In the following specific embodiments of the first binding partaccording to the invention are listed:

1. A monoclonal antibody specifically binding to BCMA, characterized incomprising a CDR3H region of SEQ ID NO:17 and a CDR3L region of SEQ IDNO:20 and a CDR1H, CDR2H, CDR1L, and CDR2L region combination selectedfrom the group of

a) CDR1H region of SEQ ID NO:21 and CDR2H region of SEQ ID NO:22, CDR1Lregion of SEQ ID NO:23, and CDR2L region of SEQ ID NO:24,

b) CDR1H region of SEQ ID NO:21 and CDR2H region of SEQ ID NO:22, CDR1Lregion of SEQ ID NO:25, and CDR2L region of SEQ ID NO:26,

c) CDR1H region of SEQ ID NO:21 and CDR2H region of SEQ ID NO:22, CDR1Lregion of SEQ ID NO:27, and CDR2L region of SEQ ID NO:28,

d) CDR1H region of SEQ ID NO:29 and CDR2H region of SEQ ID NO:30, CDR1Lregion of SEQ ID NO:31, and CDR2L region of SEQ ID NO:32,

e) CDR1H region of SEQ ID NO:34 and CDR2H region of SEQ ID NO:35, CDR1Lregion of SEQ ID NO:31, and CDR2L region of SEQ ID NO:32, and

f) CDR1H region of SEQ ID NO:36 and CDR2H region of SEQ ID NO:37, CDR1Lregion of SEQ ID NO:31, and CDR2L region of SEQ ID NO:32.

2. A monoclonal antibody specifically binding to BCMA, characterized incomprising a VH region comprising a CDR1H region of SEQ ID NO:21, aCDR2H region of SEQ ID NO:22 and a CDR3H region of SEQ ID NO:17 and a VLregion comprising a CDR3L region of SEQ ID NO:20 and a CDR1L and CDR2Lregion combination selected from the group of

a) CDR1L region of SEQ ID NO:23 and CDR2L region of SEQ ID NO:24,

b) CDR1L region of SEQ ID NO:25 and CDR2L region of SEQ ID NO:26, or

c) CDR1L region of SEQ ID NO:27 and CDR2L region of SEQ ID NO:28.

3. The antibody according to embodiment for 2, characterized incomprising as VL region a VL region selected from the group consistingof VL regions of SEQ ID NO:12, 13, and 14.

4. The antibody according to any one of embodiments 1 to 3,characterized in comprising as VH region a VH region of SEQ ID NO:10 andas VL region a VL region of SEQ ID NO:12.

5. The antibody according to any one of embodiment 1 to 3, characterizedin comprising as VH region a VH region of SEQ ID NO:10 and as VL regiona VL region of SEQ ID NO:13.

6. The antibody according to any one of embodiment 1 to 3, characterizedin comprising as VH region a

VH region of SEQ ID NO:10 and as VL region a VL region of SEQ ID NO:14.

7. The antibody according to embodiment 1 or 2, characterized in thatamino acid 49 of the VL region is selected from the group of amino acidstyrosine (Y), glutamic acid (E), serine (S), and histidine (H).

8. The antibody according to embodiment 7, characterized in that aminoacid 74 of the VL region is threonine (T) or alanine (A).

9. A monoclonal antibody specifically binding to BCMA, characterized incomprising a VH region comprising a CDR3H region of SEQ ID NO:17 and aVL region comprising a CDR1L region of SEQ ID NO:31, a CDR2L region ofSEQ ID NO:32 and a CDR3L region of SEQ ID NO:20 and a CDR1L and CDR2Lregion combination selected from the group of

a) CDR1H region of SEQ ID NO:29 and CDR2H region of SEQ ID NO:30,

b) CDR1H region of SEQ ID NO:34 and CDR2H region of SEQ ID NO:35, or

c) CDR1H region of SEQ ID NO:36 and CDR2H region of SEQ ID NO:37.

10. The antibody according to embodiment 9, characterized in comprisinga VL region of SEQ ID NO:12 and a VH region selected from the groupcomprising the VH regions of SEQ ID NO:38, 39, and 40.

11. The antibody according to embodiment 9 or 10, characterized incomprising in in that amino acid 49 of the VL region is selected fromthe group of amino acids tyrosine(Y), glutamic acid (E), serine (S), andhistidine (H).

12. The antibody according to embodiment 9 or 10, characterized in thatamino acid 74 of the VL region is threonine (T) or alanine (A).

13. The antibody according to any one of embodiments 1 to 12,characterized in that it binds also specifically to cynomolgus BCMA andcomprises an additional Fab fragment specifically binding to CD3c.

14. The antibody according to any one of embodiments 1 to 13,characterized in being an antibody with an Fc or without an Fc part.

In the following specific embodiments of the bispecific antibodyaccording to the invention the invention are listed:

15. A bispecific antibody specifically binding to BCMA and CD3c,characterized in comprising a CDR3H region of SEQ ID NO:17 and a CDR3Lregion of SEQ ID NO:20 and a CDR1H, CDR2H, CDR1L, and CDR2L regioncombination selected from the group of

a) CDR1H region of SEQ ID NO:21 and CDR2H region of SEQ ID NO:22, CDR1Lregion of SEQ ID NO:23, and CDR2L region of SEQ ID NO:24,

b) CDR1H region of SEQ ID NO:21 and CDR2H region of SEQ ID NO:22, CDR1Lregion of SEQ ID NO:25, and CDR2L region of SEQ ID NO:26,

c) CDR1H region of SEQ ID NO:21 and CDR2H region of SEQ ID NO:22, CDR1Lregion of SEQ ID NO:27, and CDR2L region of SEQ ID NO:28,

d) CDR1H region of SEQ ID NO:29 and CDR2H region of SEQ ID NO:30, CDR1Lregion of SEQ ID NO:31, and CDR2L region of SEQ ID NO:32,

e) CDR1H region of SEQ ID NO:34 and CDR2H region of SEQ ID NO:35, CDR1Lregion of SEQ ID NO:31, and CDR2L region of SEQ ID NO:32, and

f) CDR1H region of SEQ ID NO:36 and CDR2H region of SEQ ID NO:37, CDR1Lregion of SEQ ID NO:31, and CDR2L region of SEQ ID NO:32.

16. A bispecific antibody specifically binding to the two targets whichare the extracellular domain of human BCMA (further named also as“BCMA”) and human CD3ε (further named also as “CD3”), characterized incomprising a VH region comprising a CDR1H region of SEQ ID NO:21, aCDR2H region of SEQ ID NO:22 and a CDR3H region of SEQ ID NO:17 and a VLregion comprising a CDR3L region of SEQ ID NO:20 and a CDR1L and CDR2Lregion combination selected from the group of

a) CDR1L region of SEQ ID NO:23 and CDR2L region of SEQ ID NO:24,

b) CDR1L region of SEQ ID NO:25 and CDR2L region of SEQ ID NO:26, or

c) CDR1L region of SEQ ID NO:27 and CDR2L region of SEQ ID NO:28.

17. The bispecific antibody according to embodiment 15 or 16,characterized in comprising as VH region a VH region of SEQ ID NO:10.

18. The bispecific antibody according to any one of embodiments 15 to16, characterized in that the BCMA VL is selected from the groupconsisting of VL regions of SEQ ID NO:12, 13, and 14.

19. The bispecific antibody according to any one of embodiments 14 to18, characterized in comprising as BCMA VH region a VH region of SEQ IDNO:10 and as VL region a VL region of SEQ ID NO:12, or as BCMA VH a VHregion of SEQ ID NO:10 and as VL region a VL region of SEQ ID NO:13, oras BCMA VH a VH region of SEQ ID NO:10 and as VL region a VL region ofSEQ ID NO:14.

20. The bispecific antibody according to any one of embodiments 15 or19, characterized in comprising in in that amino acid 49 of the VLregion is selected from the group of amino acids tyrosine(Y), glutamicacid (E), serine (S), and histidine (H).

21. The bispecific antibody according to any one of embodiments 15 to20, characterized in that amino acid 74) of the VL region is threonine(T) or alanine (A).

22. A bispecific antibody specifically binding to BCMA and CD3,characterized in comprising a VH region comprising a CDR3H region of SEQID NO:17 and a VL region comprising a CDR1L region of SEQ ID NO:31, aCDR2L region of SEQ ID NO:32 and a CDR3L region of SEQ ID NO:20 and aCDR1L and CDR2L region combination selected from the group of

a) CDR1H region of SEQ ID NO:29 and CDR2H region of SEQ ID NO:30,

b) CDR1H region of SEQ ID NO:34 and CDR2H region of SEQ ID NO:35, or

c) CDR1H region of SEQ ID NO:36 and CDR2H region of SEQ ID NO:37.

23. The bispecific antibody according to embodiment 22, characterized incomprising a VL region of SEQ ID NO:12 and a VH region selected from thegroup comprising the VH regions of SEQ ID NO:38, 39, and 40.

24. The bispecific antibody according to embodiment 22 or 23,characterized in comprising in in that amino acid 49 of the VL region isselected from the group of amino acids tyrosine(Y), glutamic acid (E),serine (S), and histidine (H).

25. The bispecific antibody according to any one of embodiments 22 to24, characterized in that amino acid 74 of the VL region is threonine(T) or alanine (A).

26. The bispecific antibody according to any one of embodiments 15 to25, characterized in comprising an anti BCMA antibody according to theinvention and an anti CD3 antibody, wherein

a) the light chain and heavy chain of an antibody according to any oneof embodiments 1 to 7; and

b) the light chain and heavy chain of an antibody specifically bindingto CD3, wherein the variable domains VL and VH or the constant domainsCL and CH1 are replaced by each other.

27. The bispecific antibody according to any one of embodiments 15 to26, characterized in comprising not more than one Fab fragment of ananti-CD3 antibody portion, not more than two Fab fragments of ananti-BCMA antibody portion and not more than one Fc part.

28. The bispecific antibody according to any one of embodiments 15 to27, characterized in comprising a Fc part linked with its N-terminus tothe C-terminus of said CD3 antibody Fab fragment and to the C-terminusof one of said BCMA antibody Fab fragments.

29. The bispecific antibody according to any one of embodiments 15-28,characterized in comprising a second Fab fragment of said anti-BCMAantibody (BCMA antibody portion) linked with its C-terminus to theN-terminus of said Fab fragment of said anti-CD3 antibody (CD3 antibodyportion) of said bispecific antibody.

30. The bispecific antibody according to embodiment 29, characterized inthat the VL domain of said anti-CD3 antibody Fab fragment is linked tothe CH1 domain of said second anti-BCMA antibody Fab fragment.

31. The bispecific antibody according to any one of embodiments 15 to30, characterized in that the variable domain VH of the anti-CD3antibody portion (further named as “CD3 VH”) comprises the heavy chainCDRs of SEQ ID NO: 1, 2 and 3 as respectively heavy chain CDR1, CDR2 andCDR3 and the variable domain VL of the anti-CD3 antibody portion(further named as “CD3 VL”) comprises the light chain CDRs of SEQ ID NO:4, 5 and 6 as respectively light chain CDR1, CDR2 and CDR3.

32. The bispecific antibody according to any one of embodiments 15 to31, characterized in that the variable domains of the anti CD3ε antibodyportion are of SEQ ID NO:7 and 8.

33. A bispecific antibody specifically binding to the two targets whichare the extracellular domain of human BCMA and human CD3ε, characterizedin comprising

a) the first light chain and the first heavy chain of a first antibodyaccording to any one of embodiments 1 to 7; and

b) the second light chain and the second heavy chain of a secondantibody which specifically binds to CD3, and wherein the variabledomains VL and VH in the second light chain and second heavy chain ofthe second antibody are replaced by each other; and

c) wherein in the constant domain CL of the first light chain under a)the amino acid at position 124 is substituted independently by lysine(K), arginine (R) or histidine (H) (numbering according to Kabat), andwherein in the constant domain CH1 of the first heavy chain under a) theamino acid at position 147 and the amino acid at position 213 issubstituted independently by glutamic acid (E), or aspartic acid (D)(numbering according to Kabat) (see e.g. FIGS. 1A, 2A, 2C, 3A, 3C).

34. A bispecific antibody specifically according to embodiment 33,characterized in comprising in addition a Fab fragment of said firstantibody (further named also as “BCMA-Fab”) and in the constant domainCL said BCMA-Fab the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat), and wherein in the constant domain CH1 of saidBCMA-Fab the amino acid at positions 147 and the amino acid at position213 is substituted independently by glutamic acid (E), or aspartic acid(D) (numbering according to Kabat) (see e.g. FIGS. 2A, 2C).

35. A bispecific antibody specifically binding to the two targets whichare the extracellular domain of human BCMA and human CD3ε, characterizedin comprising

a) the first light chain and the first heavy chain of a first antibodyaccording to any one of embodiments 1 to 7; and

b) the second light chain and the second heavy chain of a secondantibody which specifically binds to CD3, and wherein the variabledomains VL and VH in the second light chain and second heavy chain ofthe second antibody are replaced by each other; and wherein

c) in the constant domain CL of the second light chain under b) theamino acid at position 124 is substituted independently by lysine (K),arginine (R) or histidine (H) (numbering according to Kabat), andwherein in the constant domain CH1 of the second heavy chain under b)the amino acid at positions 147 and the amino acid at position 213 issubstituted independently by glutamic acid (E), or aspartic acid (D)(numbering according to Kabat).

36. A bispecific antibody specifically binding to the two targets whichare the extracellular domain of human BCMA and human CD3ε, characterizedin comprising a heavy and light chain set selected from the groupconsisting of polypeptides

i) SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, and SEQ ID NO:51 (2×); (set1 TCB of antibody 21),

ii) SEQ ID NO:48, SEQ ID NO:52, SEQ ID NO:53, and SEQ ID NO:54 (2×) (set2 TCB of antibody 22), and

iii) SEQ ID NO:48, SEQ ID NO:55, SEQ ID NO:56, and SEQ ID NO:57 (2×)(set 3 TCB of antibody 42).

37. A chimeric antigen receptor (CAR) or the respective CAR T-cellcomprising: an antigen recognition moiety directed against BCMA and aT-cell activation moiety, characterized in that the antigen recognitionmoiety is a monoclonal antibody or antibody fragment according to anyone of embodiments 1 to 14.

38. A chimeric antigen receptor (CAR) or the respective CAR T-cellaccording to embodiment 37, characterized in comprising:

(i) a B cell maturation antigen (BCMA) recognition moiety;

(ii) a spacer domain; and

(ii) a transmembrane domain; and

(iii) an intracellular T cell signaling domain.

39. A chimeric antigen receptor (CAR) or the respective CAR T-cellaccording to embodiment 37 or 38, characterized in that the antigenrecognition moiety is a monoclonal antibody or antibody fragmentspecifically binding to BCMA, characterized in comprising a CDR3H regionof SEQ ID NO:17 and a CDR3L region of SEQ ID NO:20 and a CDR1H, CDR2H,CDR1L, and CDR2L region combination selected from the group of

a) CDR1H region of SEQ ID NO:21 and CDR2H region of SEQ ID NO:22, CDR1Lregion of SEQ ID NO:23, and CDR2L region of SEQ ID NO:24,

b) CDR1H region of SEQ ID NO:21 and CDR2H region of SEQ ID NO:22, CDR1Lregion of SEQ ID NO:25, and CDR2L region of SEQ ID NO:26,

c) CDR1H region of SEQ ID NO:21 and CDR2H region of SEQ ID NO:22, CDR1Lregion of SEQ ID NO:27, and CDR2L region of SEQ ID NO:28,

d) CDR1H region of SEQ ID NO:29 and CDR2H region of SEQ ID NO:30, CDR1Lregion of SEQ ID NO:31, and CDR2L region of SEQ ID NO:32,

e) CDR1H region of SEQ ID NO:34 and CDR2H region of SEQ ID NO:35, CDR1Lregion of SEQ ID NO:31, and CDR2L region of SEQ ID NO:32, and

f) CDR1H region of SEQ ID NO:36 and CDR2H region of SEQ ID NO:37, CDR1Lregion of SEQ ID NO:31, and CDR2L region of SEQ ID NO:32.

In one embodiment the binding of the bispecific antibody is not reducedby 100 ng/ml APRIL for more than 20% measured in an ELISA assay as OD at405 nm compared to the binding of said antibody to human BCMA withoutAPRIL, does not alter APRIL-dependent NF-κB activation for more than20%, as compared to APRIL, and does not alter NF-κB activation withoutAPRIL for more than 20%, as compared without said antibody.

In one embodiment the binding the bispecific antibody in a concentrationof 6.25 nM is not reduced by 140 ng/ml murine APRIL for more than 10%,preferably not reduced by for more than 1% measured in an ELISA assay asOD at 450 nm compared to the binding of said antibody to human BCMAwithout APRIL. The binding of said antibody in a concentration of 50 nMis not reduced by 140 ng/ml murine APRIL for more than 10%, measured inan ELISA assay as OD at 450 nm, compared to the binding of said antibodyto human BCMA without APRIL.

In one embodiment the binding of said antibody is not reduced by 100ng/ml APRIL and not reduced by 100 ng/ml BAFF for more than 20% measuredin an ELISA assay as OD at 405 nm compared to the binding of saidantibody to human BCMA without APRIL or BAFF respectively, the antibodydoes not alter APRIL-dependent NF-κB activation for more than 20%, ascompared to APRIL alone, does not alter BAFF-dependent NF-κB activationfor more than 20%, as compared to BAFF alone, and does not alter NF-κBactivation without BAFF and APRIL for more than 20%, as compared withoutsaid antibody.

In one embodiment the binding of said antibody to human BCMA is notreduced by 100 ng/ml APRIL for more than 15%, measured in said ELISA,not reduced by 1000 ng/ml APRIL, for more than 20%, measured in saidELISA, and not reduced by 1000 ng/ml APRIL for more than 15%, measuredin said ELISA.

In one embodiment the binding of said antibody to human BCMA is notreduced by 100 ng/ml APRIL and not reduced by 100 ng/ml BAFF for morethan 15%, measured in said ELISA, not reduced by 1000 ng/ml APRIL andnot reduced by 1000 ng/ml BAFF, for more than 20%, measured in saidELISA, not reduced by 1000 ng/ml APRIL and not reduced by 1000 ng/mlBAFF for more than 15%, measured in said ELISA.

In one embodiment the bispecific antibody does not alter APRIL-dependentNF-kB activation for more than 15%, does not alter BAFF-dependent NF-kBactivation for more than 15%, and does not alter NF-κB activationwithout APRIL and BAFF for more than 15%.

In one embodiment the binding of the antibody to BCMA is not reduced byAPRIL, not reduced by BAFF for more than 25%, not more than 20%, and notmore than 10%, measured as binding of said antibody in a concentrationof 5 nM, preferably 50 nM, and 140 nM to NCI-H929 cells (ATCC®CRL-9068™) in presence or absence of APRIL or respectively BAFF in aconcentration of 2.5 μg/ml compared to the binding of said antibody toNCI-H929 cells without APRIL or BAFF respectively.

In one embodiment the following examples, sequence listing and figuresare provided to aid the understanding of the present invention, the truescope of which is set forth in the appended claims. It is understoodthat modifications can be made in the procedures set forth withoutdeparting from the spirit of the invention.

Materials & General Methods

Recombinant DNA Techniques

Standard methods were used to manipulate DNA as described in Sambrook,J. et al., Molecular cloning: A laboratory manual; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989. The molecularbiological reagents were used according to the manufacturer'sinstructions. General information regarding the nucleotide sequences ofhuman immunoglobulins light and heavy chains is given in: Kabat, E. A.et al., (1991) Sequences of Proteins of Immunological Interest, 5^(th)ed., NIH Publication No. 91-3242. Amino acids of antibody chains werenumbered and referred to according to Kabat, E. A., et al., Sequences ofProteins of Immunological Interest, 5th ed., Public Health Service,National Institutes of Health, Bethesda, Md., (1991).

Gene Synthesis

a) Desired gene segments were prepared from oligonucleotides made bychemical synthesis. The 600-1800 bp long gene segments, which wereflanked by singular restriction endonuclease cleavage sites, wereassembled by annealing and ligation of oligonucleotides including PCRamplification and subsequently cloned via the indicated restrictionsites e.g. Kpnl/Sad or Ascl/Pacl into a pPCRScript (Stratagene) basedpGA4 cloning vector. The DNA sequences of the subcloned gene fragmentswere confirmed by DNA sequencing. Gene synthesis fragments were orderedaccording to given specifications at Geneart (Regensburg, Germany)

b) Desired gene segments where required were either generated by PCRusing appropriate templates or were synthesized by Geneart AG(Regensburg, Germany) from synthetic oligonucleotides and PCR productsby automated gene synthesis. The gene segments flanked by singularrestriction endonuclease cleavage sites were cloned into standardexpression vectors or into sequencing vectors for further analysis. Theplasmid DNA was purified from transformed bacteria using commerciallyavailable plasmid purification kits. Plasmid concentration wasdetermined by UV spectroscopy. The DNA sequence of the subcloned genefragments was confirmed by DNA sequencing. Gene segments were designedwith suitable restriction sites to allow sub-cloning into the respectiveexpression vectors. If required, protein coding genes were designed witha 5′-end DNA sequence coding for a leader peptide which targets proteinsfor secretion in eukaryotic cells.

DNA Sequence Determination

DNA sequences were determined by double strand sequencing.

DNA and Protein Sequence Analysis and Sequence Data Management

The Clone Manager (Scientific & Educational Software) software packageversion 9.2 was used for sequence mapping, analysis, annotation andillustration.

Expression Vectors

a) The fusion genes comprising the described antibody chains asdescribed below were generated by PCR and/or gene synthesis andassembled with known recombinant methods and techniques by connection ofthe according nucleic acid segments e.g. using unique restriction sitesin the respective vectors. The subcloned nucleic acid sequences wereverified by DNA sequencing. For transient transfections largerquantities of the plasmids are prepared by plasmid preparation fromtransformed E. coli cultures (Nucleobond AX, Macherey-Nagel).

b) For the generation of anti-BCMA antibody expression vectors, thevariable regions of heavy and light chain DNA sequences were subclonedin frame with either the human IgG1 constant heavy chain or the hum IgG1constant light chain pre-inserted into the respective generic recipientexpression vector optimized for expression in mammalian cell lines. Theantibody expression is driven by a chimeric MPSV promoter comprising aCMV enhancer and a MPSV promoter followed by a 5′ UTR, an intron and aIg kappa MAR element. The transcription is terminated by a syntheticpolyA signal sequence at the 3′ end of the CDS. All vectors carry a5′-end DNA sequence coding for a leader peptide which targets proteinsfor secretion in eukaryotic cells. In addition each vector contains anEBV OriP sequence for episomal plasmid replication in EBV EBNAexpressing cells.

c) For the generation of BCMA×CD3 bispecific antibody vectors, the IgG1derived bispecific molecules consist at least of two antigen bindingmoieties capable of binding specifically to two distinct antigenicdeterminants CD3 and BCMA. The antigen binding moieties are Fabfragments composed of a heavy and a light chain, each comprising avariable and a constant region. At least one of the Fab fragments was a“Crossfab” fragment, wherein VH and VL were exchanged. The exchange ofVH and VL within the Fab fragment assures that Fab fragments ofdifferent specificity do not have identical domain arrangements. Thebispecific molecule design was monovalent for CD3 and bivalent for BCMAwhere one Fab fragment was fused to the N-terminus of the inner CrossFab(2+1). The bispecific molecule contained an Fc part in order for themolecule to have a long half-life. A schematic representation of theconstructs is given in FIG. 2; the preferred sequences of the constructsare shown in SEQ ID NOs 39 to 52. The molecules were produced byco-transfecting HEK293 EBNA cells growing in suspension with themammalian expression vectors using polymer-based solution. Forpreparation of 2+1 CrossFab-IgG constructs, cells were transfected withthe corresponding expression vectors in a 1:2:1:1 ratio (“vectorFc(knob)”: “vector light chain”: “vector light chain CrossFab”: “vectorheavy chain-CrossFab”).

Cell Culture Techniques

Standard cell culture techniques are used as described in CurrentProtocols in Cell Biology (2000), Bonifacino, J. S., Dasso, M., Harford,J. B., Lippincott-Schwartz, J. and Yamada, K. M. (eds.), John Wiley &Sons, Inc.

Transient Expression in HEK293 Cells (HEK293-EBNA System)

Bispecific antibodies were expressed by transient co-transfection of therespective mammalian expression vectors in HEK293-EBNA cells, which werecultivated in suspension, using polymer-based solution. One day prior totransfection the HEK293-EBNA cells were seeded at 1.5 Mio viablecells/mL in Ex-Cell medium, supplemented with 6 mM of L-Glutamine. Forevery mL of final production volume 2.0 Mio viable cells werecentrifuged (5 minutes at 210×g). The supernatant was aspirated and thecells resuspended in 100 μL of CD CHO medium. The DNA for every mL offinal production volume was prepared by mixing 1 μg of DNA (Ratio heavychain: modified heavy chain: light chain: modified light chain=1:1:2:1)in 100 μL of CD CHO medium. After addition of 0.27 μL of polymer-basedsolution (1 mg/mL) the mixture was vortexed for 15 seconds and left atroom temperature for 10 minutes. After 10 minutes, the resuspended cellsand DNA/polymer-based solution mixture were put together and thentransferred into an appropriate container which was placed in a shakingdevice (37° C., 5% CO₂). After a 3 hours incubation time 800 μL ofEx-Cell Medium, supplemented with 6 mM L-Glutamine, 1.25 mM valproicacid and 12.5% Pepsoy (50 g/L), was added for every mL of finalProduction volume. After 24 hours, 70 μL of feed solution was added forevery mL of final production volume. After 7 days or when the cellviability was equal or lower than 70%, the cells were separated from thesupernatant by centrifugation and sterile filtration. The antibodieswere purified by an affinity step and one or two polishing steps, beingcation exchange chromatography and size exclusion chromatography. Whenrequired, an additional polishing step was used. The recombinantanti-BCMA human antibody and bispecific antibodies were produced insuspension by co-transfecting HEK293-EBNA cells with the mammalianexpression vectors using polymer-based solution. The cells weretransfected with two or four vectors, depending in the format. For thehuman IgG1 one plasmid encoded the heavy chain and the other plasmid thelight chain. For the bispecific antibodies four plasmids wereco-transfected. Two of them encoded the two different heavy chains andthe other two encoded the two different light chains. One day prior totransfection the HEK293-EBNA cells were seeded at 1.5 Mio viablecells/mL in F17 Medium, supplemented with 6 mM of L-Glutamine.

Protein Determination

Determination of the antibody concentration was done by measurement ofthe absorbance at 280 nm, using the theoretical value of the absorbanceof a 0.1% solution of the antibody. This value was based on the aminoacid sequence and calculated by GPMAW software (Lighthouse data).

SDS-PAGE

The NuPAGE® Pre-Cast gel system (Invitrogen) is used according to themanufacturer's instruction. In particular, 10% or 4-12% NuPAGE® Novex®Bis-TRIS Pre-Cast gels (pH 6.4) and a NuPAGE® MES (reduced gels, withNuPAGE® Antioxidant running buffer additive) or MOPS (non-reduced gels)running buffer is used.

Protein Purification

By Protein a Affinity Chromatography

For the affinity step the supernatant was loaded on a protein A column(HiTrap Protein A FF, 5 mL, GE Healthcare) equilibrated with 6 CV 20 mMsodium phosphate, 20 mM sodium citrate, pH 7.5. After a washing stepwith the same buffer the antibody was eluted from the column by stepelution with 20 mM sodium phosphate, 100 mM sodium chloride, 100 mMGlycine, pH 3.0. The fractions with the desired antibody wereimmediately neutralized by 0.5 M Sodium Phosphate, pH 8.0 (1:10), pooledand concentrated by centrifugation. The concentrate was sterile filteredand processed further by cation exchange chromatography and/or sizeexclusion chromatography.

By Cation Exchange Chromatography

For the cation exchange chromatography step the concentrated protein wasdiluted 1:10 with the elution buffer used for the affinity step andloaded onto a cation exchange column (Poros 50 HS, Applied Biosystems).After two washing steps with the equilibration buffer and a washingbuffer resp. 20 mM sodium phosphate, 20 mM sodium citrate, 20 mM TRIS,pH 5.0 and 20 mM sodium phosphate, 20 mM sodium citrate, 20 mM TRIS, 100mM sodium chloride pH 5.0 the protein was eluted with a gradient using20 mM sodium phosphate, 20 mM sodium citrate, 20 mM TRIS, 100 mM sodiumchloride pH 8.5. The fractions containing the desired antibody werepooled, concentrated by centrifugation, sterile filtered and processedfurther a size exclusion step.

By Analytical Size Exclusion Chromatography

For the size exclusion step the concentrated protein was injected in aXK16/60 HiLoad Superdex 200 column (GE Healthcare), and 20 mM Histidine,140 mM Sodium Chloride, pH 6.0 with or without Tween20 as formulationbuffer. The fractions containing the monomers were pooled, concentratedby centrifugation and sterile filtered into a sterile vial.

Measurement of Purity and Monomer Content

Purity and monomer content of the final protein preparation wasdetermined by CE-SDS (Caliper LabChip GXII system (Caliper LifeSciences)) resp. HPLC (TSKgel G3000 SW XL analytical size exclusioncolumn (Tosoh)) in a 25 mM potassium phosphate, 125 mM Sodium chloride,200 mM L-arginine monohydrochloride, 0.02% (w/v) Sodium azide, pH 6.7buffer.

Molecular Weight Confirmation by LC-MS Analyses

Deglycosylation

To confirm homogeneous preparation of the molecules final proteinsolution of was analyzed by LC-MS analyses. To remove heterogeneityintroduced by carbohydrates the constructs are treated with PNGaseF(ProZyme). Therefore the pH of the protein solution was adjusted topH7.0 by adding 2 μl 2 M Tris to 20 μg protein with a concentration of0.5 mg/ml. 0.8 μg PNGaseF was added and incubated for 12 h at 37° C.

LC-MS Analysis—on Line Detection

The LC-MS method was performed on an Agilent HPLC 1200 coupled to a TOF6441 mass spectrometer (Agilent). The chromatographic separation wasperformed on a Macherey Nagel Polysterene column; RP1000-8 (8 μmparticle size, 4.6×250 mm; cat. No. 719510). Eluent A was 5%acetonitrile and 0.05% (v/v) formic acid in water, eluent B was 95%acetonitrile, 5% water and 0.05% formic acid. The flow rate was 1ml/min, the separation was performed at 40° C. and 6 μg (15 μl) of aprotein sample obtained with a treatment as described before.

Time (min.) % B 0.5 15 10 60 12.5 100 14.5 100 14.6 15 16 15 16.1 100

During the first 4 minutes the eluate was directed into the waste toprotect the mass spectrometer from salt contamination. The ESI-sourcewas running with a drying gas flow of 12 l/min, a temperature of 350° C.and a nebulizer pressure of 60 psi. The MS spectra were acquired using afragmentor voltage of 380 V and a mass range 700 to 3200 m/z in positiveion mode using. MS data were acquired by the instrument software from 4to 17 minutes.

Isolation of Human PBMCs Blood

Peripheral blood mononuclear cells (PBMCs) were prepared by Histopaquedensity centrifugation from enriched lymphocyte preparations (huffycoats) obtained from local blood banks or from fresh blood from healthyhuman donors. Briefly, blood was diluted with sterile PBS and carefullylayered over a Histopaque gradient (Sigma, H8889). After centrifugationfor 30 minutes at 450×g at room temperature (brake switched off), partof the plasma above the PBMC containing interphase was discarded. ThePBMCs were transferred into new 50 ml Falcon tubes and tubes were filledup with PBS to a total volume of 50 ml. The mixture was centrifuged atroom temperature for 10 minutes at 400×g (brake switched on). Thesupernatant was discarded and the PBMC pellet washed twice with sterilePBS (centrifugation steps at 4° C. for 10 minutes at 350×g). Theresulting PBMC population was counted automatically (ViCell) and storedin RPMI1640 medium, containing 10% FCS and 1% L-alanyl-L-glutamine(Biochrom, K0302) at 37° C., 5% CO₂ in the incubator until assay start.

Isolation of Primary Cynomolgus PBMCs from Heparinized Blood

Peripheral blood mononuclear cells (PBMCs) were prepared by densitycentrifugation from fresh blood from healthy cynomolgus donors, asfollows: Heparinized blood was diluted 1:3 with sterile PBS, andLymphoprep medium (Axon Lab #1114545) was diluted to 90% with sterilePBS. Two volumes of the diluted blood were layered over one volume ofthe diluted density gradient and the PBMC fraction was separated bycentrifugation for 30 min at 520×g, without brake, at room temperature.The PBMC band was transferred into a fresh 50 ml Falcon tube and washedwith sterile PBS by centrifugation for 10 min at 400×g at 4° C. Onelow-speed centrifugation was performed to remove the platelets (15 minat 150×g, 4° C.), and the resulting PBMC population was automaticallycounted (ViCell) and immediately used for further assays.

EXAMPLES Example 1: Generation of Anti-BCMA Antibodies Example 1.1:Production of Antigens and Tool Reagents Example 1.1.1: Recombinant,Soluble, Human BCMA Extracellular Domain

The extracellular domains of human, cynomolgus and murine BCMA that wereused as antigens for phage display selections were transiently expressedas N-terminal monomeric Fc-fusion in HEK EBNA cells and in vivosite-specifically biotinylated via co-expression of BirA biotin ligaseat the avi-tag recognition sequence located at the C-terminus of the Fcportion carrying the receptor chain (Fc knob chain). The extracellulardomains of human and cynomolgus BCMA comprised methionine 4 toasparagine 53, and methionine 4 to asparagine 52, respectively. Thesewere N-terminally fused to the hinge of a human IgG1 enablingheterodimerization with an unfused human IgG1 Fc portion (hole chain) byknobs-into-holes technology.

Example 1.1.1 Generation of Anti-BCMA Antibodies by Maturation

1.1.1A.1 Libraries and Selections

Two libraries were constructed on basis of antibody 83A10. Theselibraries are randomized in either CDR1 and CDR2 of the light chain(83A10 L1/L2) or CDR1 and CDR2 of the heavy chain (83A10 L1/L2),respectively. Each of these libraries was constructed by 2 subsequentsteps of amplification and assembly. Final assembly products have beendigested NcoI/BsiWI for 83A10 L1/L2 library, MunI and NheI for 83A10H1/H2 library, alongside with similarly treated acceptor vectors basedon plasmid preparations of clone 83A10. The following amounts ofdigested randomized (partial) V-domains and digested acceptor vector(s)were ligated for the respective libraries (μg V-domain/μg vector):a.m.83A10 L1/L2 library (3/10), 83A10 H1/H2 library (3/10), Purifiedligations of 83A10 L1/L2 and 83A10 H1/H2 libraries were pooled,respectively, and used for 15 transformations of E.coli TG1 cells foreach of the 2 libraries, to obtain final library sizes of 2.44×10¹⁰ for83A10 L1/L2 library, and 1.4×10¹⁰ for a.m.83A10 H1/H2 library. Phagemidparticles displaying these Fab libraries were rescued and purified.

1.1.1A.2 Selections of Clones Selections were carried out against theectodomain of human or cyno B-cell maturation antigen (BCMA) to whichwas cloned upstream a Fc and an avi-tag. Prior to selections, the Fcdepleter was coated onto neutravidin plates at a concentration of 500nM. Selections were carried out according to the following pattern:

-   -   1) binding of ˜10¹² phagemid particles of library.83A10 L1/L2        library or 83A10 HI/H2 library to immobilized Fc depleter for        1 h. 2) transfer of unbound phagemid particles of library.83A 10        L1/L2 library or 83A 10 H1/H2 library to 50 nM, 25 nM, 10 nM, or        2 nM human or cyno BCMA (depending on library and selection        round) for 20 min. 3) adding magnetic streptavidin beads for 10        min. 4) washing of magnetic streptavidin beads using 10×1 ml        PBS/Tween® 20 and 10×1 ml PBS, 5) elution of phage particles by        addition of 1 ml 100 mM TEA (triethylamine) for 10 min and        neutralization by addition of 500 ul 1M Tris®/HCl pH 7.4 and 6)        re-infection of log-phase E. coli TG1 cells, infection with        helper phage VCSM13 and subsequent PEG/NaCl precipitation of        phagemid particles to be used in subsequent selection rounds.

Selections have been carried out over 3 rounds and conditions wereadjusted in 5 streamlines for each of the 2 libraries individually. Indetail, selection parameters were:

Streamline 1 (50 nM huBCMA for round 1, 25 nM cynoBCMA for round 2, 10nM huBCMA for round 3),

Streamline 2 (50 nM huBCMA for round 1, 10 nM huBCMA for round 2, 2 nMhuBCMA for round 3).

Streamline 3 (50 nM huBCMA for round 1, 25 nM huBCMA for round 2, 10 nMcynoBCMA for round 3).

Streamline 4 (50 nM huBCMA for round 1, 25 nM cynoBCMA for round 2, 10nM cynoBCMA for round 3),

Streamline 5 (50 nM cynoBCMA for round 1, 25 nM cynoBCMA for round 2, 10nM cynoBCMA for round 3).

The heavy chains of Mab 21, Mab 22, Mab 33, and Mab 42 BCMA antibodieswere derived from Streamline 5 which used only cynoBCMA.

1.1.1A.3 Screening Method

Individual clones were bacterially expressed as 1 ml cultures in 96-wellformat and supernatants were subjected to a screening by ELISA. Specificbinders were defined as signals higher than 5× background for human andcyno BCMA and signals lower than 3× background for Fc depleter.Neutravidin 96 well strip plates were coated with 10 nM of huBCMA, 10 nMcyBCMA or 50 nM Fe-depleter followed by addition of Fab-containingbacterial supernatants and detection of specifically binding Fabs viatheir Flag-tags by using an anti-Flag/HRP secondary antibody.ELISA-positive clones were bacterially expressed as 1 ml cultures in96-well format and supernatants were subjected to a kinetic screeningexperiment ProteOn. 500 positive clones were identified, most of themhaving similar affinity. 1.1.1A.4 Surface Plasmon Resonance Screen withSoluble Fabs and IgGs

70 clones were further tested by SPR. All experiments were performed at25° C. using PBST as running buffer (10 mM PBS, pH 7.4 and 0.005% (v/v)Tween®20). A ProteOn XPR36 biosensor equipped with GLC and GLM sensorchips and coupling reagents (10 mM sodium acetate, pH 4.5.sulfo-N-hydroxysuccinimide,1-ethyl-3-(3-dimethylaminpropyl)-carbodiimide hydrochloride [EDC] andethanolamine) was purchased from BioRad Inc. (Hercules, Calif.).Immobilizations were performed at 30 μl/min on a GLM chip. pAb (goat)anti hu IgG, F(ab)2 specific Ab (Jackson) was coupled in verticaldirection using a standard amine-coupling procedure: all six ligandchannels were activated for 5 min with a mixture of EDC (200 mM) andsulfo-NHS (50 mM). Immediately after the surfaces were activated. pAb(goat) anti hu IgG, F(ab)2 specific antibody (50 μg/ml, 10 mM sodiumacetate, pH 5) was injected across all six channels for 5 min. Finally,channels were blocked with a 5 min injection of 1 M ethanolamine-HCl (pH8.5). Final immobilization levels were similar on all channels, rangingfrom 11000 to 11500 RU. The Fab variants were captured from E.colisupernatants by simultaneous injection along five of the separate wholehorizontal channels (30 μl/min) for 5 min and resulted in levels,ranging from 200 to 900 RU, depending on the concentration of Fab insupernatant; conditioned medium was injected along the sixth channel toprovide an ‘in-line’ blank for double referencing purposes. One-shotkinetic measurements were performed by injection of a dilution series ofhuman. cyno and mouse BCMA (50, 10, 2, 0.4, 0.08, 0 nM, 50 μl/min) for 3min along the vertical channels. Dissociation was monitored for 5 min.Kinetic data were analyzed in ProteOn Manager v. 2.1. Processing of thereaction spot data involved applying an interspot-reference and adouble-reference step using an inline buffer blank (Myszka, 1999). Theprocessed data from replicate one-shot injections were fit to a simple1:1 Langmuir binding model without mass transport (O'Shannessy et al.,1993).

For measurements of IgG from supernatants of HEK productions in 6-wellformat, the IgG variants were captured from HEK293 supernatants bysimultaneous injection along five of the separate whole horizontalchannels (30 μl/min) for 5 min and resulted in levels, ranging from 200to 400 RU; conditioned medium was injected along the sixth channel toprovide an ‘in-line’ blank for double referencing purposes. One-shotkinetic measurements were performed by injection of a dilution series ofhuman, cyno and mouse BCMA (25, 5, 1, 0.2, 0.04, 0 nM, 50 μl/min) for 3min along the vertical channels. Dissociation was monitored for 5 min.Kinetic data were analyzed as described above. The OSK measurements aresummarized in Table 2D; i/m, inconclusive measurement. Affinity tohuBCMA was found to be between about 50 μm to 5 nM. Affinity to cynoBCMAwas found to be between about 2 nM to 20 nM (few clones fall outside therange, see FIG. 17).

1.1.1A5. Further Selection of HC and LC Clones

Due to their experience the inventors selected out of these 70 clonesfurther 27 clones based on their binding properties to huBCMA, cynoBCMA,murineBCMA, and ratio, measured in different assays. Out of these clones4VH and 9VL clones were selected, which results in 34 VH/VLcombinations. Binding affinity on HEK-huBCMA cells was measured (FIG. 18and Table 2E). It was found that binding of antibodies Mab 21, Mab 22,Mab 27, Mab 39 and Mab 42 to huBCMA on HEK cells was not significantlybetter than the binding of Mab 83A10 to huBCMA-HEK cells. However Mab21,Mab 22, Mab27, Mab33, Mab39, and Mab42 were selected due to theiroverall properties, like affinity for huBCMA, cynoBCMA, binding asbispecific antibody to BCMA-positive multiple myeloma cell lines H929,L363 and RPMI-8226 by flow cytometry, killing potency of myeloma cellsH929, L363 and RPMI-8226, of viable myeloma plasma cells from patientbone marrow aspirates, and pharmacokinetics (PK)) and pharmacodynamics(killing of BCMA positive cells) data in cynomolgus monkeys.

TABLE 2C Relationship of antibodies to streamlines Derived from Derivedfrom Mab No. library 2 (HC) Clone HC library 1 (LC) Clone LC Mab 21Streamline 5 5F04 Streamline 1 1D04 Mab 22 Streamline 5 5F04 Streamline1 1C05 Mab 27 Streamline 1 1A08 Streamline 1 1D04 Mab 33 Streamline 55D03 Streamline 1 1D04 Mab 39 Streamline 2 2E12 Streamline 1 1D04 Mab 42Streamline 5 5F04 Streamline 5 5A11

TABLE 2D One-shot-kinetic affinity measurements to human, cynomolgus andmouse BCMA Mab KD KD KD No. VH VL huBCMA cyBCMA muBCMA 83A10 pCON1532pCON1080 1.5E−09 1.4E−08 i/m Mab 21 pCON1531 pCON1522 2.8E−11 5.1E−117.3E−10 Mab 22 pCON1531 pCON1521 4.8E−11 i/m 9.0E−10 Mab 27 pCON1520pCON1522 3.9E−13 1.0E−10 9.7E−10 Mab 33 pCON1530 pCON1522 1.7E−113.4E−11 4.9E−10 Mab 39 pCON1524 pCON1522 6.2E−11 2.7E−10 i/m Mab 42pCON1531 pCON1527 2.3E−10 3.9E−10 2.5E−09

TABLE 2E Binding of IgG variants on HEK-huBCMA cells Binding Binding MabNo VH VL EC50 [nM] EC50 [μg/mL] 83A10 PCON1532 PCON1080 2.4 0.34 Mab 14PCON1530 PCON1527 1.47 0.21 Mab 21 pCON1531 PCON1522 2.46 0.35 Mab 22PCON1531 pCON1521 2.08 0.30 Mab 23 PCON1531 PCON1519 4.97 0.71 Mab 27PCON1520 PCOM1522 10.57 1.52 Mab 28 PCON1520 PCOM1521 11.34 1.63 Mab 30PCON1530 PCON1526 10.35 1.49 Mab 31 PCON1530 PCON1525 1.34 0.19 Mab 33pCOM1530 PCON1522 1.18 0.17 Mab 34 PCON1530 PCON1521 1.24 0.18 Mab 35PCON1530 PCON1519 1.63 0.23 Mab 39 PCON1524 PCON1522 1.73 0.25 Mab 42PCON1531 pCON1527 2.10 0.30 Mab 44 PCON1520 PCON1527 1.55 0.22

Example 1.2: BCMA-Expressing Cells as Tools Example 1.2.1: Human MyelomaCell Lines Expressing BCMA on their Surface and Quantification of BCMAReceptor Number on Cell Surface

BCMA expression was assessed on five human myeloma cell lines (NCI-H929,RPMI-8226, U266B1, L-363 and JJN-3) by flow cytometry. NCI-H929 cells((H929) ATCC® CRL-9068™) were cultured in 80-90% RPMI 1640 with 10-20%heat-inactivated FCS and could contain 2 mM L-glutamine, 1 mM sodiumpyruvate and 50 μM mercaptoethanol. RPMI-8226 cells ((RPMI) ATCC®CCL-155™) were cultured in a media containing 90% RPMI 1640 and 10%heat-inactivated FCS. U266B1 ((U266) ATCC® TIB-196™) cells were culturedin RPMI-1640 medium modified to contain 2 mM L-glutamine, 10 mM HEPES, 1mM sodium pyruvate, 4500 mg/L glucose, and 1500 mg/L sodium bicarbonateand 15% heat-inactivated FCS. L-363 cell line (Leibniz InstituteDSMZ—German collection of microorganisms and cell cultures; DSMZ No. ACC49) was cultured in 85% RPMI 1640 and 15% heat-inactivated FCS. JJN-3cell line (DSMZ No. ACC 541) was cultured in 40% Dulbecco's MEM+40%Iscove's MDM+20% heat-inactivated FBS. Briefly, cells were harvested,washed, counted for viability, resuspended at 50,000 cells/well of a96-well round bottom plate and incubated with anti-human BCMA antibody(Abcam, #ab54834, mouse IgG1) at 10 μg/ml for 30 min at 4° C. (toprevent internalization). A mouse IgG1 was used as isotype control (BDBiosciences, #554121). Cells were then centrifuged (5 min at 350×g),washed twice and incubated with the FITC-conjugated anti mouse secondaryantibody for 30 min at 4° C. At the end of incubation time, cells werecentrifuged (5 min at 350×g), washed twice with FACS buffer, resuspendedin 100 ul FACS buffer and analyzed on a Cantoll device running FACS Divasoftware. The relative quantification of BCMA receptor number on thesurface membrane of H929, RPMI-8226 and U266B1 myeloma cell lines wasassessed by QIFIKIT analysis (Dako, #K0078, following manufacturer'sinstructions). H929 cells expressed human BCMA with the highest density,up to 5-6-fold higher more than other myeloma cell lines. H929 isconsidered as a high BCMA-expressing myeloma cell line as compared toU266 and L363 which are medium/low BCMA-expressing myeloma cells,RPMI-8226 which are low BCMA-expressing myeloma cells and JJN-3 whichare very low BCMA-expressing myeloma cells. Table 3 summarizes therelative BCMA receptor number on the cell surface of human multiplemyeloma cell lines per each experiment (n=5).

TABLE 3 Quantification of BCMA receptor number on membrane surface ofH929, L363, RPMI-8226, U266B1 and JJN-3 human myeloma cell lines HumanSpecific antigen binding capacity (SABC) myeloma Exper- Exper- Exper-Exper- Exper- cell lines iment 1 iment 2 iment 3 iment 4 iment 5 H92919357 54981 44800 100353  98050  L363 16,970  / 11300 11228 / U266(B1) /12852 11757 / 9030 RPMI-8226  1165  5461 / 11361 2072 JJN-3 / / / /  650

Example 2: BCMA Binding Assays: Surface Plasmon Resonance

Assessment of binding of anti-BCMA antibodies to recombinant BCMA bysurface plasmon resonance (SPR) as follow. All SPR experiments wereperformed on a Biacore T200 at 25° C. with HBS-EP as running buffer(0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20,Biacore, Freiburg/Germany). The avidity of the interaction betweenanti-BCMA antibodies and recombinant BCMA Fc(kih) (human and cynomolgus)was determined. Biotinylated recombinant human and cynomolgus BCMAFc(kih) were directly coupled on a SA chip following instructions(Biacore, Freiburg/Germany). The immobilization level ranged from 200 to700 RU. The anti-BCMA antibodies were passed at a 2-fold concentrationrange (1.95 to 500 nM) with a flow of 30 μL/minutes through the flowcells over 120 seconds. The dissociation was monitored for 180 seconds.Bulk refractive index differences were corrected for by subtracting theresponse obtained on the reference flow cell. Here, the anti-BCMAantibodies were flown over an empty surface previously activated anddeactivated as described in the standard amine coupling kit. Apparentkinetic constants were derived using the Biacore T200 EvaluationSoftware (vAA, Biacore AB, Uppsala/Sweden), to fit rate equations for1:1 Langmuir binding by numerical integration, despite the bivalency ofthe interaction for comparison purposes. The affinity of the interactionbetween anti-BCMA antibodies and recombinant human BCMA Fc(kih) was alsodetermined. Anti-human Fab antibody (GE Healthcare) was directly coupledon a CMS chip at pH 5.0 using the standard amine coupling kit (Biacore,Freiburg/Germany). The immobilization level was about 6500 RU. Anti-BCMAantibody was captured for 90 seconds at 25 nM. Recombinant human BCMAFc(kih) was passed at a 4-fold concentration range (1.95 to 500 nM) witha flow of 30 μL/minutes through the flow cells over 120 seconds. Thedissociation was monitored for 120 seconds. Bulk refractive indexdifferences were corrected for by subtracting the response obtained onreference flow cell. Here, recombinant BCMA was flown over a surfacewith immobilized anti-human Fab antibody but on which HBS-EP has beeninjected rather than anti-BCMA antibody. Kinetic constants were derivedusing the Biacore T100 Evaluation Software (vAA, Biacore AB,Uppsala/Sweden), to fit rate equations for 1:1 Langmuir binding bynumerical integration (Table 4).

TABLE 4 Affinity constants determined by fitting rate equations for 1:1Langmuir binding Ligand Analyte Kon[1/Ms] Koff[1/s] KD[M] 83A10 IgGhuBCMA Fc(kih) 5.07E+05 2.92E−03 5.76E−09 cynoBCMA Fc(kih) 2.29E+052.03E−02 8.86E−08 Mab 21 IgG huBCMA Fc(kih) 8.51E+05 4.39E−05 5.16E−11cynoBCMA Fc(kih) 4.91E+05 2.35E−04 4.78E−10 Mab 22 IgG huBCMA Fc(kih)8.14E+05 5.15E−05 6.33E−11 cynoBCMA Fc(kih) 4.54E+05 4.42E−04 9.74E−10Mab 42 IgG huBCMA Fc(kih) 8.03E+05 2.98E−04 3.71E−10 cynoBCMA Fc(kih)7.07E+05 4.53E−04 6.41E−10 Mab 27 IgG huBCMA Fc(kih) 3.59E+05 5.93E−051.65E−10 cynoBCMA Fc(kih) 2.16E+05 4.55E−04 2.11E−09 Mab 33 IgG huBCMAFc(kih) 2.00E+05 3.55E−05 1.78E−10 cynoBCMA Fc(kih) 1.32E+05 9.76E−057.39E−10 Mab 39 IgG huBCMA Fc(kih) 3.61E+05 5.58E−05 1.55E−10 cynoBCMAFc(kih) 2.15E+05 4.67E−04 2.17E−09

Example 3: Human/Cynomolgus (Hu/Cyno) Affinity Gap

Based on the affinity values described in Example 2, the affinity ofanti-BCMA antibodies to human BCMA vs. cynomolgus BCMA were compared andcyno/hu affinity ratio (gap) values were calculated (Table 5). Affinitycyno/hu gap was calculated as affinity of antibody to cynomolgus BCMAdivided by affinity to human BCMA and means that BCMA antibody binds tohuman BCMA with x fold binding affinity than to cynomolgus BCMA, wherex=cyno/hu gap value. Results are shown in Table 5.

TABLE 5 Affinity of anti-BCMA antibodies to human BCMA vs. cynomolgusBCMA and hu/cyno gap values K_(D) human KD cynomolgus Affinity α-BCMAIgG BCMA[M] BCMA[M] cyno/hu gap 83A10 5.76E−09 8.86E−08 15.3 Mab 215.16E−11 4.78E−10 9.3 Mab 22 6.33E−11 9.74E−10 15.4 Mab 42 3.71E−106.41E−10 1.7 Mab 27 1.65E−10 2.11E−09 12.7 Mab 33 1.78E−10 7.39E−10 4.2Mab 39 1.55E−10 2.17E−09 14

Example 4: Generation of Anti-BCMA/Anti-CD3 T Cell Bispecific Antibodies

Anti-BCMA/anti-CD3 T cell bispecific antibodies were generated accordingto WO2014/122144, which is incorporated by reference.

Example 4.1: Anti-CD3 Antibodies

The term “CD3ε or CD3” as used herein relates to human CD3ε describedunder UniProt P07766 (CD3E_HUMAN). The term “antibody against CD3, antiCD3 antibody” relates to an antibody binding to CD3ε. Preferably theantibody comprises a variable domain VH comprising the heavy chain CDRsof SEQ ID NO: 1, 2 and 3 as respectively heavy chain CDR1, CDR2 and CDR3and a variable domain VL comprising the light chain CDRs of SEQ ID NO:4, 5 and 6 as respectively light chain CDR1, CDR2 and CDR3. Preferablythe antibody comprises the variable domains of SEQ ID NO:7 (VH) and SEQID NO:8 (VL). Anti-CD3 antibody as described above was used to generatethe T cell bispecific antibodies which were used in the followingexamples,

Example 4.2: Generation of Anti-BCMA/Anti-CD3 T Cell BispecificAntibodies of Fc-Containing 2+1 Format

cDNAs encoding the full heavy and light chains of the correspondinganti-BCMA IgG1 antibodies as well as the anti-CD3 VH and VL cDNAs wereused as the starting materials. For each bispecific antibody, fourprotein chains were involved comprising the heavy and light chains ofthe corresponding anti-BCMA antibody and the heavy and light chains ofthe anti-CD3 antibody described above, respectively. In order tominimize the formation of side-products with mispaired heavy chains, forexample with two heavy chains of the anti-CD3 antibody, a mutatedheterodimeric Fc region is used carrying “knob-into-hole mutations” andan engineered disulphide bond, as described in WO2009080251 and inWO2009080252. In order to minimize the formation of side-products withmispaired light chains, for example with two light chains of theanti-BCMA antibody, a CH1× constant kappa crossover is applied to theheavy and light chains of the anti-CD3 antibody using the methodologydescribed in WO2009080251 and in WO2009080252.

a) An anti-BCMA/anti-CD3 T cell bispecific antibody with a 2+1 formati.e. bispecific (Fab)₂× (Fab) antibody that is bivalent for BCMA andmonovalent for CD3 would have advantages on potency, predictability forefficacy and safety because it would preferentially bind to the tumortarget BCMA and avoid CD3 antibody sink, thus higher probability fordrug exposure focused to the tumor.

Anti-BCMA/anti-CD3 T cell bispecific of the 2+1 format (i.e. bispecific(Fab)₂× (Fab) antibody bivalent for BCMA and monovalent for CD3 with Fcwere produced for the human BCMA antibodies previously selected. cDNAsencoding the full Fabs (heavy chain VH and CH1 domains plus light chainVL and CL domains) of the corresponding anti-BCMA IgG1 antibodies aswell as the anti-CD3 VH and VL cDNAs, were used as the startingmaterials. For each bispecific antibody, four protein chains wereinvolved comprising the heavy and light chains of the correspondinganti-BCMA antibody and the heavy and light chains of the anti-CD3antibody described above, respectively, with Fc regions.

Briefly, each bispecific antibody is produced by simultaneouscotransfection of four mammalian expression vectors encoding,respectively: a) the full light chain cDNA of the corresponding BCMAantibody, b) a fusion cDNA generated by standard molecular biologymethods, such as splice-overlap-extension PCR, encoding a fusion proteinmade of (in N- to C-terminal order) secretory leader sequence, Fab (VHfollowed by CH1 domains) of the corresponding anti-BCMA antibodydescribed above, a flexible glycine(Gly)-serine(Ser) linker with thesequence Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser, Fab (VH followed byCH1 domains) of the corresponding anti-BCMA antibody described above, aflexible glycine(Gly)-serine(Ser) linker with the sequenceGly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser, the VH of the anti-CD3 antibodydescribed above and the constant kappa domain of a human light chaincDNA, c) a fusion cDNA generated by standard molecular biology methods,such as splice-overlap-extension PC, encoding a fusion protein made of(in N- to C-terminal order) secretory leader sequence, VL of theanti-CD3 antibody described above, constant CH1 domain of a human IgG1cDNA. Co-transfection of mammalian cells and antibody production andpurification using the methods described above for production of humanor humanized IgG1 antibodies, with one modification: for purification ofantibodies, the first capture step is not done using ProteinA, butinstead is done using an affinity chromatography column packed with aresin binding to human kappa light chain constant region, such asKappaSelect (GE Healthcare Life Sciences). In addition, a disulfide canbe included to increase the stability and yields as well as additionalresidues forming ionic bridges and increasing the heterodimerizationyields (EP 1870459A1).

For the generation of BCMA×CD3 bispecific antibody vectors, the IgG1derived bispecific molecules consist at least of two antigen bindingmoieties capable of binding specifically to two distinct antigenicdeterminants CD3 and BCMA. The antigen binding moieties were Fabfragments composed of a heavy and a light chain, each comprising avariable and a constant region. At least one of the Fab fragments was a“Crossfab” fragment, wherein the constant domains of the Fab heavy andlight chain were exchanged. The exchange of heavy and light chainconstant domains within the Fab fragment assures that Fab fragments ofdifferent specificity do not have identical domain arrangements andconsequently do not interchange light chains. The bispecific moleculedesign was monovalent for CD3 and bivalent for BCMA where one Fabfragment is fused to the N-terminus of the inner CrossFab (2+1). Thebispecific molecule contained an Fc part in order to have a longerhalf-life. A schematic representation of the constructs is given inFIGS. 1-3; the sequences of the preferred constructs are shown in Table2A. The molecules were produced by co-transfecting HEK293 EBNA cellsgrowing in suspension with the mammalian expression vectors usingpolymer-based solution. For preparation of 2+1 CrossFab-IgG constructs,cells were transfected with the corresponding expression vectors in a1:2:1:1 ratio (“vector Fc(knob)”: “vector light chain”: “vector lightchain CrossFab”: “vector heavy chain-CrossFab”).

Example 4.3: Generation of Anti-BCMA/Anti-CD3 T Cell BispecificAntibodies for Comparison

The generation of BCMA50-sc(Fv)₂ (also known as BCMA50-BiTE®)anti-BCMA/anti-CD3 T cell bispecific antibody and the amino acidsequences used were according to WO2013072406 and WO2013072415.

Example 5: Production and Purification of Anti-BCMA/Anti-CD3Fc-Containing (2+1) T Cell Bispecific Antibodies with Charge Variants

Anti-BCMA/anti-CD3 T cell bispecific antibodies were produced andpurified according to WO2014/122144, which is incorporated by reference.

For the production of the bispecific antibodies, bispecific antibodieswere expressed by transient co-transfection of the respective mammalianexpression vectors in HEK293-EBNA cells, which were cultivated insuspension, using polymer-based solution. One day prior to transfectionthe HEK293-EBNA cells were seeded at 1.5 Mio viable cells/mL in Ex-Cellmedium, supplemented with 6 mM of L-Glutamine. For every mL of finalproduction volume 2.0 Mio viable cells were centrifuged (5 minutes at210×g). The supernatant was aspirated and the cells resuspended in 100μL of CD CHO medium. The DNA for every mL of final production volume wasprepared by mixing 1 μg of DNA (Ratio heavy chain: modified heavy chain:light chain: modified light chain=1:1:2:1) in 100 μL of CD CHO medium.After addition of 0.27 μL of polymer-based solution (1 mg/mL) themixture was vortexed for 15 seconds and left at room temperature for 10minutes. After 10 minutes, the resuspended cells and DNA/polymer-basedsolution mixture were put together and then transferred into anappropriate container which was placed in a shaking device (37° C., 5%CO₂). After a 3 hours incubation time 800 μL of Ex-Cell Medium,supplemented with 6 mM L-Glutamine, 1.25 mM valproic acid and 12.5%Pepsoy (50 g/L), was added for every mL of final Production volume.After 24 hours, 70 μL of feed solution was added for every mL of finalproduction volume. After 7 days or when the cell viability was equal orlower than 70%, the cells were separated from the supernatant bycentrifugation and sterile filtration. The antibodies were purified byan affinity step and one or two polishing steps, being cation exchangechromatography and size exclusion chromatography. When required, anadditional polishing step was used.

For the affinity step the supernatant was loaded on a protein A column(HiTrap Protein A FF, 5 mL, GE Healthcare) equilibrated with 6 CV 20 mMsodium phosphate, 20 mM sodium citrate, pH 7.5. After a washing stepwith the same buffer the antibody was eluted from the column by stepelution with 20 mM sodium phosphate, 100 mM sodium chloride, 100 mMGlycine, pH 3.0. The fractions with the desired antibody wereimmediately neutralized by 0.5 M Sodium Phosphate, pH 8.0 (1:10), pooledand concentrated by centrifugation. The concentrate was sterile filteredand processed further by cation exchange chromatography and/or sizeexclusion chromatography.

For the cation exchange chromatography step the concentrated protein wasdiluted 1:10 with the elution buffer used for the affinity step andloaded onto a cation exchange column (Poros 50 HS, Applied Biosystems).After two washing steps with the equilibration buffer and a washingbuffer resp. 20 mM sodium phosphate, 20 mM sodium citrate, 20 mM TRIS,pH 5.0 and 20 mM sodium phosphate, 20 mM sodium citrate, 20 mM TRIS, 100mM sodium chloride pH 5.0 the protein was eluted with a gradient using20 mM sodium phosphate, 20 mM sodium citrate, 20 mM TRIS, 100 mM sodiumchloride pH 8.5. The fractions containing the desired antibody werepooled, concentrated by centrifugation, sterile filtered and processedfurther a size exclusion step.

For the size exclusion step the concentrated protein was injected in aXK16/60 HiLoad Superdex 200 column (GE Healthcare), and 20 mM Histidine,140 mM Sodium Chloride, pH 6.0 with or without Tween20 as formulationbuffer. The fractions containing the monomers were pooled, concentratedby centrifugation and sterile filtered into a sterile vial.

Determination of the antibody concentration was done by measurement ofthe absorbance at 280 nm, using the theoretical value of the absorbanceof a 0.1% solution of the antibody. This value was based on the aminoacid sequence and calculated by GPMAW software (Lighthouse data).

Purity and monomer content of the final protein preparation wasdetermined by CE-SDS (Caliper LabChip GXII system (Caliper LifeSciences)) resp. HPLC (TSKgel G3000 SW XL analytical size exclusioncolumn (Tosoh)) in a 25 mM potassium phosphate, 125 mM Sodium chloride,200 mM L-arginine monohydrochloride, 0.02% (w/v) Sodium azide, pH 6.7buffer.

To verify the molecular weight of the final protein preparations andconfirm the homogeneous preparation of the molecules final proteinsolution, liquid chromatography-mass spectometry (LC-MS) was used. Adeglycosylation step was first performed. To remove heterogeneityintroduced by carbohydrates, the constructs were treated with PNGaseF(ProZyme). Therefore, the pH of the protein solution was adjusted topH7.0 by adding 2 μl 2 M Tris to 20 μg protein with a concentration of0.5 mg/ml. 0.8 μg PNGaseF was added and incubated for 12 h at 37° C. TheLC-MS online detection was then performed. LC-MS method was performed onan Agilent HPLC 1200 coupled to a TOF 6441 mass spectrometer (Agilent).The chromatographic separation was performed on a Macherey NagelPolysterene column; RP1000-8 (8 μm particle size, 4.6×250 mm; cat. No.719510). Eluent A was 5% acetonitrile and 0.05% (v/v) formic acid inwater, eluent B was 95% acetonitrile, 5% water and 0.05% formic acid.The flow rate was 1 ml/min, the separation was performed at 40° C. and 6μg (15 μl) of a protein sample obtained with a treatment as describedbefore.

During the first 4 minutes, the eluate was directed into the waste toprotect the mass spectrometer from salt contamination. The ESI-sourcewas running with a drying gas flow of 12 l/min, a temperature of 350° C.and a nebulizer pressure of 60 psi. The MS spectra were acquired using afragmentor voltage of 380 V and a mass range 700 to 3200 m/z in positiveion mode using. MS data were acquired by the instrument software from 4to 17 minutes.

FIG. 10 of EP14179705 (incorporated by reference) depicts the CE-SDS(non-reduced) graphs of the final protein preparations after differentmethods of purification for 83A10-TCB and 83A10-TCBcv antibodies.Protein A (PA) affinity chromatography and size exclusionchromatographic (SEC) purification steps applied to 83A10-TCB antibodyresulted in a purity of <30% and 82.8% of monomer content (A). Whenadditional purifications steps including cation exchange chromatography(cIEX) and a final size exclusion chromatographic (re-SEC) steps wereapplied to the final protein preparations in (A), the purity wasincreased to 93.4% but the monomer content remained the same and theyield was significantly reduced to 0.42 mg/L. However, when specificcharge modifications were applied to 83A10 anti-BCMA Fab CL-CH1, namely83A10-TCBcv antibody, a superior production/purification profile of theTCB molecule, as demonstrated by a purity of 95.3%, monomer content of100% and yield of up to 3.3 mg/L, could already be observed even whenPA+cIEX+SEC purification steps were applied (C) in comparison to (B)with a production/purification profile showing a 7.9-fold lower yieldand 17.2% lower monomer content despite including an additional re-SECpurification step.

A head-to-head production run to compare the production/purificationprofile of 83A10-TCB vs. 83A10-TCBcv antibodies was then conducted tofurther evaluate the advantages of the CL-CH1 charge modificationsapplied to the antibodies. 83A10-TCB and 83A10-TCBcv molecules are bothof molecular format as described in FIG. 2a . As depicted in FIG. 11,properties of 83A10-TCB and 83A10-TCBcv antibodies were measuredside-by-side and compared after each purification steps 1) PA affinitychromatography only (A, B), 2) PA affinity chromatography then SEC (C,D) and 3) PA affinity chromatography then SEC then cIEX and re-SEC (E,F). The CE-SDS (non-reduced) graphs of the final protein solutions afterthe respective methods of purification for 83A10-TCB and 83A10-TCBcvantibodies are demonstrated in FIG. 11 of EP14179705 (incorporated byreference). As shown in FIGS. 11A and 11B of EP14179705 (incorporated byreference), improvements with applying the charge variants to the TCBantibody were already observed after purification by PA affinitychromatography only. In this head-to-head study, PA affinitychromatography purification step applied to 83A10-TCB antibody resultedin a purity of 61.3%, a yield of 26.2 mg/L and 63.7% of monomer content(11A). In comparison, when 83A10-TCBcv antibody was purified by PAaffinity chromatography all the properties were improved with a betterpurity of 81.0%, a better yield of 51.5 mg/L and 68.2% of monomercontent (11B). When an additional SEC purification step was applied tothe final protein preparations as seen in FIGS. 12A and 12B ofEP14179705 (incorporated by reference), 83A10-TCB gained a purity of69.5%, a yield of 14.1 mg/L and 74.7% of monomer content (C) as comparedto 83A10-TCBcv with improved purity and monomer content of up to 91.0%and 83.9% respectively, and a yield of 10.3 mg/L (D). Even though theyield was slightly less (i.e. 27% less) for 83A10-TCBcv than for83A10-TCB in this particular experiment, the percentage of correctmolecule was much better for 83A10-TCBcv than for 83A10-TCB,respectively 90% vs. 40-60%, as measured by LC-MS. In the thirdhead-to-head comparison, 83A10-TCB and 83A10-TCBcv final proteinpreparations from FIGS. 11C and 11D of EP14179705 (incorporated byreference) were pooled with approximately 1 L (equivolume) of respectivefinal protein preparations from another purification batch (sameproduction) following PA affinity chromatography purification step only.The pooled protein preparations were then being further purified by cIEXand SEC purification methods. As depicted in FIGS. 11E and 11F ofEP14179705 (incorporated by reference), improvement of theproduction/purification profile of the TCB antibody with the chargevariants was consistently observed when compared to TCB antibody withoutcharge variant. After several steps of purification methods (i.e.PA+/−SEC+cIEX+SEC) were used to purify 83A10-TCB antibody, only 43.1%purity was reached and 98.3% of monomer content could be achieved but tothe detriment of the yield which was reduced to 0.43 mg/L. Thepercentage of correct molecule as measured by LC-MS was still poor with60-70%. At the end, the quality of the final protein preparation was notacceptable for in vitro use. In stark contrast, when the same multiplepurification steps with the same chronology were applied to 83A10-TCBcvantibody, 96.2% purity and 98.9% of monomer content were reached as wellas 95% of correct molecule as measured by LC-MS. The yield however wasalso greatly reduced to 0.64 mg/L after cIEX purification step. Theresults show that better purity, higher monomer content, higherpercentage of correct molecule and better yield can be achieved with83A10-TCBcv antibody only after two standard purification steps i.e. PAaffinity chromatography and SEC (FIG. 11D of EP14179705) while suchproperties could not be achieved with 83A10-TCB even when additionalpurification steps were applied (FIG. 11E of EP14179705).

Table 12 of EP14179705 (incorporated by reference) summarizes theproperties of 83A10-TCB as compared to 83A10-TCVcv following PApurification step. Table 13 of EP14179705 (incorporated by reference)summarizes the properties of 83A10-TCB as compared to 83A10-TCVcvfollowing PA and SEC purification steps. Table 14 of EP14179705(incorporated by reference) summarizes the properties of 83A10-TCB ascompared to 83A10-TCVcv following PA and SEC plus PA alone then cIEX andre-SEC purification steps. For Tables 12 to 14 of EP14179705(incorporated by reference), the values in bold highlight the superiorproperty as compared between 83A10-TCB vs. 83A10-TCVcv. With oneexception (i.e. yield respectively amount, see Table 13 of EP14179705(incorporated by reference)) which may not be representative, all theproduction/purification parameters and values resulting from the 3head-to-head comparison experiments were superior for 83A10-TCBcv ascompared to 83A10-TCB. The overall results clearly demonstrate thatadvantages in production/purification features could be achieved withapplying CL-CH1 charge modifications to TCB antibodies and that only twopurification steps (i.e PA affinity chromatography and SEC) wererequired to achieve already high quality protein preparations with verygood developability properties. Based on the improvedproduction/purification properties of 83A10-TCBcv, 21-TCBcv, 22-TCBcv,27-TCBcv, 33-TCBcv, 39-TCBcv and 42-TCBcv were generated with chargevariants, in a similar way as 83A10-TCBcv.

TABLE 6 Production/purification profile of anti-BCMA/anti- CD3 T cellbispecific antibodies following protein A affinity chromatographypurification step 83A10-TCB 83A10-TCBcv Purity (%) 61.3 81.0 Yield(mg/L) 26.2 51.5 Amount (mg) 24.3 50.2 Monomer (%) 63.7 68.2 Correctmolecule by n.d. n.d LC-MS (%)

TABLE 7 Production/purification profile of anti-BCMA/anti-CD3 T cellbispecific antibodies following protein A affinity chromatography andsize exclusion chromatography purification steps 83A10-TCB 83A10-TCBcvPurity (%) 69.5 91.0 Yield (mg/L) 14.1 10.3 Amount (mg) 13.1 10.0Monomer (%) 74.7 83.9 Correct molecule by 40-60 90 LC-MS (%)

TABLE 8 Production/purification profile of anti-BCMA/anti-CD3 T cellbispecific antibodies following 1.a) protein A affinity chromatographyand size exclusion chromatography and 1.b) protein A affinitychromatography only pooled together then 2) cation exchangechromatography and 3) final size exclusion chromatography purificationsteps 83A10-TCB 83A10-TCBcv Purity (%) 43.1 96.2 Yield (mg/L) 0.43 0.64Amount (mg) 0.73 1.27 Monomer (%) 98.3 98.9 Correct molecule by 60-70%>95% LC-MS (%)

Example 6: Binding of Anti-BCMA/Anti-CD3 T-Cell Bispecific Antibodies toBCMA-Positive Multiple Myeloma Cell Lines (Flow Cytometry)

Anti-BCMA/anti-CD3 TCB antibodies (21-TCBcv, 22-TCBcv, 42-TCBcv,83A10-TCBcv) were analyzed by flow cytometry for binding to human BCMAon BCMA-expressing H929, L363 and RPMI-8226 cells. MKN45 (human gastricadenocarcinoma cell line that does not express BCMA) was used asnegative control. Briefly, cultured cells are harvested, counted andcell viability was evaluated using ViCell. Viable cells are thenadjusted to 2×10⁶ cells per ml in BSA-containing FACS Stain Buffer (BDBiosciences). 100 μl of this cell suspension were further aliquoted perwell into a round-bottom 96-well plate and incubated with 30 μl of theanti-BCMA antibodies or corresponding IgG control for 30 min at 4° c.All Anti-BCMA/anti-CD3 TCB antibodies (and TCB controls) were titratedand analyzed in final concentration range between 1-300 nM. Cells werethen centrifuged (5 min, 350×g), washed with 120 μl/well FACS StainBuffer (BD Biosciences), resuspended and incubated for an additional 30min at 4° C. with fluorochrome-conjugated PE-conjugated AffiniPureF(ab′)2 Fragment goat anti-human IgG Fc Fragment Specific (JacksonImmuno Research Lab; 109-116-170). Cells were then washed twice withStain Buffer (BD Biosciences), fixed using 100 ul BD Fixation buffer perwell (#BD Biosciences, 554655) at 4° C. for 20 min, resuspended in 120μl FACS buffer and analyzed using BD FACS Cantoll. When applicable, EC50were calculated using Prism GraphPad (LaJolla, Calif., USA) and EC50values denoting the antibody concentration required to reaching 50% ofthe maximal binding for the binding of anti-BCMA/anti-CD3 TCB antibodiesto H929 cells, L363 cells and RPMI-8226 cells are summarized in Table 8,Table 9, and Table 10 respectively. Asterix denotes estimated EC50values as extrapolated and calculated by Prism software. EC50 values forbinding of 21-TCBcv to L363 cells and binding of 22-TCBcv to RPMI-8226cells could not be estimated

TABLE 8 EC50 values for binding of anti-BCMA/anti-CD3 T-cell bispecificantibodies to H929 multiple myeloma cells Estimated EC50 83A10-TCBcv21-TCBcv 22-TCBcv 42-TCBcv nM 12.0 11.0 7.9 13.6 μg/ml 1.725 1.589 1.1421.956

TABLE 9 EC50 values for binding of anti-BCMA/anti-CD3 T-cell bispecificantibodies to L363 multiple myeloma cells Estimated EC50 83A10-TCBcv21-TCBcv 22-TCBcv 42-TCBcv nM 17.4 / 30.0 3.8 μg/ml 2.507 / 4.328 0.5534

TABLE 10 EC50 values for binding of anti-BCMA/anti-CD3 T-cell bispecificantibodies to RPMI-8226 multiple myeloma cells Estimated EC5083A10-TCBcv 21-TCBcv 22-TCBcv 42-TCBcv nM ~188428* 6.8 / 13.2 μg/ml ~27151* 0.9817 / 1.907

Example 7: Cytokine Production from Activated T Cells Upon Binding ofAnti-BCMA/Anti-CD3 T Cell Bispecific Antibodies to CD3-Positive T Cellsand BCMA-Positive Multiple Myeloma Cell Lines (Cytokine Release AssayCBA Analysis)

Anti-BCMA/anti-CD3 T cell bispecific antibodies are analyzed for theirability to induce T-cell mediated cytokine production de novo in thepresence or absence of human BCMA-expressing human myeloma cells(RPMI-8226, JJN-3). Briefly, human PBMCs are isolated from Buffy Coatsand 0.3 million cells per well are plated into a round-bottom 96-wellplate. Alternatively, 280 μl whole blood from a healthy donor are platedper well of a deep-well 96-well plate. BCMA-positive tumor target cellsare added to obtain a final E:T-ratio of 10:1. Anti-BCMA/anti-CD3 TCBantibodies and controls are added for a final concentration of 0.1 pM-10nM. After an incubation of up to 24 h at 37° C., 5% CO₂, the assay plateis centrifuged for 5 min at 350×g and the supernatant is transferredinto a new deep-well 96-well plate for the subsequent analysis. The CBAanalysis was performed on FACS Cantoll according to manufacturer'sinstructions, using either the Human Th1/Th2 Cytokine Kit II (BD#551809) or the combination of the following CBA Flex Sets: humangranzyme B (BD #560304), human IFN-γ Flex Set (BD #558269), human TNF-αFlex Set (BD #558273), human IL-10 Flex Set (BD #558274), human IL-6Flex Set (BD #558276), human IL-4 Flex Set (BD #558272), human IL-2 FlexSet (BD #558270). Table 13 shows that 83A10-TCBcv induced aconcentration-dependent increase in cytokine production and serineprotease granzyme B, a marker of cytotoxic T-cell function. Table 11shows the EC50 values and amount of secreted cytokines/proteases peranti-BCMA/anti-CD3 T-cell bispecific antibody concentrations.

TABLE 11 Secretion of cytokine and proteases induced by anti-BCMA/anti-CD3 T-cell bispecific antibodies in presence of RPMI-8226 cellsCytokines/ EC50 83A10-TCBcv concentration (nM) proteases (nM) 0.000640.0032 0.016 0.08 0.4 2 10 TNF-α 0.52 −6.95 −6.49 −0.65 46.72 161.24315.11 371.47 (pg/mL) IL-10 0.30 −9.21 1.95 25.17 125.82 401.42 602.64680.05 (pg/mL) Granzyme B 0.34 220.54 331.55 889.13 5855.02 15862.8421270.43 27120.52 (pg/mL)

Example 8: Redirected T-Cell Cytotoxicity of BCMA-High Expressing H929Myeloma Cells Induced by Anti-BCMA/Anti-CD3 T Cell Bispecific Antibodies(Colorimetric LDH Release Assay)

Anti-BCMA/anti-CD3 TCB antibodies were analyzed for their potential toinduce T cell-mediated apoptosis in BCMA high-expressing MM cells uponcrosslinking of the construct via binding of the antigen bindingmoieties to BCMA on cells. Briefly, human BCMA high-expressing H929multiple myeloma target cells were harvested with Cell DissociationBuffer, washed and resuspended in RPMI supplemented with 10% fetalbovine serum (Invitrogen). Approximately, 30,000 cells per well wereplated in a round-bottom 96-well plate and the respective dilution ofthe construct was added for a desired final concentration (intriplicates); final concentrations ranging from 0.1 pM to 10 nM. For anappropriate comparison, all TCB constructs and controls were adjusted tothe same molarity. Human PBMCs (effector cells) were added into thewells to obtain a final E:T ratio of 10:1, corresponding to a E:T ratioof approximately 3 to 5 T cells for 1 tumor target cells. Negativecontrol groups were represented by effector or target cells only. Fornormalization, maximal lysis of the H929 MM target cells (=100%) wasdetermined by incubation of the target cells with a final concentrationof 1% Triton X-100, inducing cell death. Minimal lysis (=0%) wasrepresented by target cells co-incubated with effector cells only, i.e.without any T cell bispecific antibody. After 20-24h or 48h incubationat 37° C., 5% CO₂, LDH release from the apoptotic/necrotic MM targetcells into the supernatant was then measured with the LDH detection kit(Roche Applied Science), following the manufacturer's instructions. Thepercentage of LDH release was plotted against the concentrations ofanti-BCMA/anti-CD3 T cell bispecific antibodies inconcentration-response curves. The EC50 values were measured using Prismsoftware (GraphPad) and determined as the TCB antibody concentrationthat results in 50% of maximum LDH release. As shown in FIG. 4, allanti-BCMA/anti-CD3 TCB antibodies (21-, 22-, 42-, and 83A10-TCBcv)induced a concentration-dependent killing of BCMA-positive H929 myelomacells as measured by LDH release. The lysis of H929 cells was specificsince control-TCB antibody which does not bind to BCMA-positive targetcells but only to CD3 on T cells did not induce LDH release, even at thehighest concentration tested. Table 12 summarizes the EC50 values forthe redirected T-cell killing of BCMA high-expressing H929 cells inducedby anti-BCMA/anti-CD3 TCB antibodies.

TABLE 12 EC50 values for redirected T-cell killing of H929 cells inducedby anti-BCMA/anti-CD3 TCB antibodies Anti-BCMA/ anti-CD3 EC50 (pM) TCBantibodies Donor 1 Donor 2 Donor 3 Donor 4 Donor 5 Donor 6 21-TCBcv 97.1/ 42.1 53.9 38.7 / 22-TCBcv 53.2 / 42.2 23.2 28.9 / 42-TCBcv 9.7 / 11.77.2 6.8 / 83A10-TCBcv 3.9 / 8.5 5.0 4.3 1.5

Example 9: Redirected T-Cell Cytotoxicity of BCMA-Medium/Low ExpressingL363 Myeloma Cells Induced by Anti-BCMA/Anti-CD3 T Cell BispecificAntibodies (LDH Release Assay)

Anti-BCMA/anti-CD3 TCB antibodies were also analyzed for their abilityto induce T cell-mediated apoptosis in BCMA medium/low-expressing MMcells upon crosslinking of the construct via binding of the antigenbinding moieties to BCMA on cells. Briefly, human BCMAmedium/low-expressing L363 multiple myeloma target cells are harvestedwith Cell Dissociation Buffer, washed and resuspended in RPMIsupplemented with 10% fetal bovine serum (Invitrogen). Approximately,30,000 cells per well are plated in a round-bottom 96-well plate and therespective dilution of the construct is added for a desired finalconcentration (in triplicates); final concentrations ranging from 0.1 pMto 10 nM. For an appropriate comparison, all TCB constructs and controlsare adjusted to the same molarity. Human PBMCs (effector cells) wereadded into the wells to obtain a final E:T ratio of 10:1, correspondingto a E:T ratio of approximately 3 to 5 T cells for 1 tumor target cells.Negative control groups were represented by effector or target cellsonly. For normalization, maximal lysis of the MM target cells (=100%) isdetermined by incubation of the target cells with a final concentrationof 1% Triton X-100, inducing cell death Minimal lysis (=0%) wasrepresented by target cells co-incubated with effector cells only, i.e.without any T cell bispecific antibody. After 20-24 h incubation at 37°C., 5% CO₂, LDH release from the apoptotic/necrotic MM target cells intothe supernatant was then measured with the LDH detection kit (RocheApplied Science), following the manufacturer's instructions. Thepercentage of LDH release was plotted against the concentrations ofanti-BCMA/anti-CD3 T cell bispecific antibodies inconcentration-response curves. The EC50 values were measured using Prismsoftware (GraphPad) and determined as the TCB antibody concentrationthat results in 50% of maximum LDH release. As shown in FIG. 5, allanti-BCMA/anti-CD3 TCB antibodies (21-, 22-, 42-, and 83A10-TCBcv)induced a concentration-dependent killing of BCMA-positive L363 myelomacells as measured by LDH release. The lysis of L363 cells was specificsince control-TCB antibody which does not bind to BCMA-positive targetcells but only to CD3 on T cells did not induce LDH release, even at thehighest concentration tested. Table 13 summarizes the EC50 values forthe redirected T-cell killing of BCMA medium/low-expressing L363 cellsinduced by anti-BCMA/anti-CD3 TCB antibodies.

TABLE 13 EC50 values for redirected T-cell killing of L363 cells inducedby anti-BCMA/anti-CD3 TCB antibodies Anti-BCMA/anti-CD3 EC50 (pM) TCBantibodies Donor 1 Donor 2 Donor 3 Donor 4 Donor 5 21-TCBcv 83.6 38.418.9 19.1 46.4 22-TCBcv 97.5 27.7 16.5 14.6 56.0 42-TCBcv 15.5 16.7 5.22.2 10.6 83A10-TCBcv 16.8 47.8 28.4 12.6 39.0

Example 10: Redirected T-Cell Cytotoxicity of BCMA-Medium/Low ExpressingRPMI-8226 Myeloma Cells Induced by Anti-BCMA/Anti-CD3 T Cell BispecificAntibodies (LDH Release Assay)

Anti-BCMA/anti-CD3 TCB antibodies were analyzed for their ability toinduce T cell-mediated apoptosis in BCMA medium/low-expressing MM cellsupon crosslinking of the construct via binding of the antigen bindingmoieties to BCMA on cells. Briefly, human BCMA medium/low-expressingL363 multiple myeloma target cells are harvested with Cell DissociationBuffer, washed and resuspended in RPMI supplemented with 10% fetalbovine serum (Invitrogen). Approximately, 30,000 cells per well areplated in a round-bottom 96-well plate and the respective dilution ofthe construct is added for a desired final concentration (intriplicates); final concentrations ranging from 0.1 pM to 10 nM. For anappropriate comparison, all TCB constructs and controls are adjusted tothe same molarity. Human PBMCs (effector cells) were added into thewells to obtain a final E:T ratio of 10:1, corresponding to a E:T ratioof approximately 3 to 5 T cells for 1 tumor target cells. Negativecontrol groups were represented by effector or target cells only. Fornormalization, maximal lysis of the MM target cells (=100%) wasdetermined by incubation of the target cells with a final concentrationof 1% Triton X-100, inducing cell death Minimal lysis (=0%) wasrepresented by target cells co-incubated with effector cells only, i.e.without any T cell bispecific antibody. After 20-24 h incubation at 37°C., 5% CO₂, LDH release from the apoptotic/necrotic MM target cells intothe supernatant was then measured with the LDH detection kit (RocheApplied Science), following the manufacturer's instructions. Thepercentage of LDH release was plotted against the concentrations ofanti-BCMA/anti-CD3 T cell bispecific antibodies inconcentration-response curves. The EC50 values were measured using Prismsoftware (GraphPad) and determined as the TCB antibody concentrationthat results in 50% of maximum LDH release. As shown in FIG. 6, allanti-BCMA/anti-CD3 TCB antibodies (21-, 22-, 42-, and 83A10-TCBcv)induced a concentration-dependent killing of BCMA-positive RPMI-8226myeloma cells as measured by LDH release. The lysis of RPMI-8226 cellswas specific since control-TCB antibody which does not bind toBCMA-positive target cells but only to CD3 on T cells did not induce LDHrelease, even at the highest concentration tested. Table 13 summarizesthe EC50 values for the redirected T-cell killing of BCMAmedium/low-expressing RPMI-8226 cells induced by anti-BCMA/anti-CD3 TCBantibodies.

TABLE 13 EC50 values for redirected T-cell killing of RPMI-8226 cellsinduced by anti-BCMA/anti-CD3 TCB antibodies Anti-BCMA/anti-CD3 EC50(pM) TCB antibodies Donor 1 Donor 2 Donor 3 Donor 4 Donor 5 21-TCBcv /41.3 8.8 4.0 8.4 22-TCBcv / 47.6 7.6 3.2 5.5 42-TCBcv / 382.8 18.7 3.51.5 83A10-TCBcv / 620.5 229.3 35.0 64.9

Example 11: Redirected T-Cell Cytotoxicity of BCMA-Low Expressing JJN-3Myeloma Cells Induced by Anti-BCMA/Anti-CD3 T Cell Bispecific Antibodies(Flow Cytometry and LDH Release)

Anti-BCMA/anti-CD3 TCB antibodies were analyzed for their ability toinduce T cell-mediated apoptosis in BCMA low-expressing MM cells uponcrosslinking of the construct via binding of the antigen bindingmoieties to BCMA on cells. Briefly, human BCMA low-expressing JJN-3multiple myeloma target cells are harvested with Cell DissociationBuffer, washed and resuspended in RPMI supplemented with 10% fetalbovine serum (Invitrogen). Approximately, 30,000 cells per well areplated in a round-bottom 96-well plate and the respective dilution ofthe construct is added for a desired final concentration (intriplicates); final concentrations ranging from 0.1 pM to 10 nM. For anappropriate comparison, all TCB constructs and controls are adjusted tothe same molarity. Human PBMCs (effector cells) were added into thewells to obtain a final E:T ratio of 10:1, corresponding to a E:T ratioof approximately 3 to 5 T cells for 1 tumor target cells. Negativecontrol groups were represented by effector or target cells only. Fornormalization, maximal lysis of the MM target cells (=100%) wasdetermined by incubation of the target cells with a final concentrationof 1% Triton X-100, inducing cell death Minimal lysis (=0%) wasrepresented by target cells co-incubated with effector cells only, i.e.without any T cell bispecific antibody. i) After 48 h incubation at 37°C., 5% CO₂, the cultured myeloma cells were collected, washed andstained with fluorochrome-conjugated antibodies and Annexin-V fordetermination of apoptotic myeloma cells. The staining panel comprisedCD138-APCC750/CD38-FITC/CD5-BV510/CD56-PE/CD19-PerCP-Cy7/CD45-V450/Annexin-V-PerCP-Cy5.5.Fluorochrome-labelled antibodies used were purchased from BD Biosciences(San Jose, Calif.) and Caltag Laboratories (San Francisco Calif.).Acquisition was performed using a multicolor flow cytometer andinstalled software (e.g. Cantoll device running FACS Diva software orFACSCalibur flow cytometer using the CellQUEST software). ThePaint-A-Gate PRO program (BD Biosciences) was used for data analysis.Annexin-V was measured on JJN-3 cells and the percentage ofannexin-v-positive JJN-3 cells was plotted against the concentration ofanti-BCMA/anti-CD3 T cell bispecific antibodies. The percentage of lysisof JJN-3 cells induced by a specific concentration of anti-BCMA/anti-CD3T cell bispecific antibody was also determined by measuring the absolutecount of annexin-V-negative JJN-3 cells at a given TCB concentration andsubtracting it from the absolute count of annexin-V-negative JJN-3 cellswithout TCB; divided by the absolute count of annexin-V-negative JJN-3cells without TCB. FIG. 7 shows that anti-BCMA/anti-CD3 TCB antibodies(22-, 42-, and 83A10-TCBcv) induced a concentration-dependent killing ofBCMA low-expressing JJN-3 myeloma cells as measured by flow cytometry.The lysis of JJN-3 cells was specific since control-TCB antibody whichdoes not bind to BCMA-positive target cells but only to CD3 on T cellsdid not induce increase in annexin-v positive JJN-3 cells or JJN-3 celllysis, even at the highest concentration tested. Table 14 and Table 15summarize respectively the percentages of annexin-v positive JJN-3 cellsand percentages of lysis of JJN-3 cells induced by anti-BCMA/anti-CD3TCB antibodies.

Detection of LDH is also performed after 20-24 h or 48 h incubation at37° C., 5% CO₂. LDH release from the apoptotic/necrotic JJN-3 MM targetcells into the supernatant is then measured with the LDH detection kit(Roche Applied Science), following the manufacturer's instructions. Thepercentage of LDH release is plotted against the concentrations ofanti-BCMA/anti-CD3 T cell bispecific antibodies inconcentration-response curves. The EC50 values are measured using Prismsoftware (GraphPad) and determined as the TCB antibody concentrationthat results in 50% of maximum LDH release.

TABLE 14 Redirected T-cell killing of BCMA low-expressing JJN-3 cellsinduced by anti-BCMA/anti-CD3 TCB antibodies: percentages of annexin-Vpositive cells Annexin-V positive Anti-BCMA/anti-CD3 TCB concentration(pM) JJN-3 cells (%) 10000 1000 100 10 1 0.1 0 Experiment 1 83A10-TCBcv16.78 10.21 9.12 11.11 11.36  8.14 9.6 42-TCBcv 24.83 16.84 8.62 12.3 11.9 / 9.6 22-TCBcv 22.95 26.15 12.48 13.29 9.3 12.48 9.6 Control-TCB8.84 / / / / / / Experiment 2 83A10-TCBcv 22.86 17.53 16.5 15.94 14.3213.07 10.74 42-TCBcv 26.88 21.68 14.42 13.6  13.47 12.75 10.74 22-TCBcv29.72 26.97 18.35 15.94 15 14.8  10.74 Control-TCB 12.82 / / / / / /

TABLE 15 Redirected T-cell killing of BCMA low-expressing JJN-3 cellsinduced by anti-BCMA/anti-CD3 TCB antibodies: percentages of lysis ofJJN-3 cells Lysis of Anti-BCMA/anti-CD3 TCB concentration (pM) JJN-3cells (%) 10000 1000 100 10 1 0.1 0 Experiment 1 83A10-TCBcv 70.30 26.6618.43 41.88 24.42 −14.45  0.00 42-TCBcv 92.92 84.02 41.87 38.96 40.29 /0.00 22-TCBcv 88.02 90.54 56.26 73.56 −4.29 26.28 0.00 Control-TCB −6.55/ / / / / / Experiment 2 83A10-TCBcv 51.18 25.30 20.12 39.58 −1.88 22.280.00 42-TCBcv 90.37 81.12 55.32 39.44 34.94 17.62 0.00 22-TCBcv 91.2194.12 53.03 41.66 24.36 36.47 0.00 Control-TCB 4.18 / / / / / /

Example 12: BCMA Expression on Bone Marrow Myeloma Plasma Cells fromMultiple Myeloma Patients

Human cell lines expressing the tumor target of interest are very usefuland practical tools for the measurement of TCB antibody potency toinduce tumor cell cytotoxicity in presence of T cells and determinationof EC50 values and for the ranking of TCB molecules. However, despitebeing readily accessible and practical human myeloma cell lines have thecaveat of not representing the heterogeneity of multiple myeloma, a verycomplex disease which is characterized by a significant heterogeneity atthe molecular level. In addition, myeloma cell lines do not express BCMAreceptor with the same intensity and density as some cells express BCMAmore strongly than others (e.g. H929 cells vs. RPMI-8226 cells), andsuch heterogeneity at the cellular level may also be observed amongdifferent patients. Throughout academic collaborations with key opinionleaders in multiple myeloma, determination of BCMA expression anddensity in patient samples and evaluation of the anti-BCMA/anti-CD3 TCBantibodies with clinical patient samples are being investigated. Bloodand bone marrow aspirates are collected from multiple myeloma patientsafter informed consent is given, in accordance with local ethicalcommittee guidelines and the Declaration of Helsinki.

a) BCMA Expression as Detected by Multiparameter Flow Cytometry (MeanFluorescence Intensity)

To determine the expression of BCMA receptor on bone marrow myelomacells, immunophenotypic analyses were performed using freshly isolatedwhole bone marrow aspirates. Erythrocyte-lysed K₃-EDTA(ethylenediaminetetraacetic acid) anticoagulated whole bone marrowsamples were used for the immunophenotypic analyses. A total of 2×10⁶cells per tube were stained, lysed, and then washed using a directimmunofluorescence technique and multicolor staining, which was aimed atthe specific identification and immunophenotypic characterization ofmalignant plasma cells identified as CD138⁺ CD38⁺ CD45⁺ CD19⁻ CD56⁺. Thecells were then stained using a panel of fluorochrome-conjugatedantibodies including at leastCD38-FITC/CD56-PE/CD19-PerCP-Cy7/CD45-V450/BCMA-APC.Fluorochrome-labelled antibodies used are purchased from BD Biosciences(San Jose, Calif.) and Caltag Laboratories (San Francisco Calif.).In-house APC-conjugated anti-human BCMA antibody was used in theimmunophenotypic analyses. Acquisition was performed using a multicolorflow cytometer and installed software (e.g. Cantoll device running FACSDiva software or FACSCalibur flow cytometer using the CellQUESTsoftware). The Paint-A-Gate PRO program (BD Biosciences) was used fordata analysis. BCMA expression was measured gated on the malignantplasma cell population and mean fluorescence intensity (MFI) values weredetermined and compared among the myeloma patients.

TABLE 16 BCMA expression on patient bone marrow myeloma plasma cells asdetected by multiparameter flow cytometry (mean fluorescence intensity)Patient No MFI_(BCMA) P1 2863 P2 3528 P3 602 P4 389 P5 955 P6 1475 P7282 P8 1621 P9 116 P10 125 P11 1495 P12 2451 P13 398 P14 2040 P15 678P16 945 P17 1672 P18 1491 P19 2198 P20 1058 P21 3594 P22 615 P23 159

b) Determination of BCMA Specific Antigen Binding Capacity (QuantitativeFlow Cytometry Analysis)

The Qifikit (Dako) method was used to quantify BCMA specific antigenbinding capacity (SABC) on the cell surface of patient bone marrowmyeloma plasma cells. Myeloma plasma cells isolated from whole bonemarrow aspirates were stained with 50 μl of mouse anti-human BCMA IgG(BioLegend #357502) or a mouse IgG2a isotype control (BioLegend #401501)diluted in FACS buffer (PBS, 0.1% BSA) to a final concentration of 25μg/ml (or at saturation concentrations) and staining was performed for30 min at 4° C. in the dark. Next, 100 μl of the Set-up or CalibrationBeads were added in separate wells and the cells, as well as the beadswere washed twice with FACS buffer. Cells and beads were resuspended in25 μl FACS buffer, containing fluorescein conjugated anti-mousesecondary antibody (at saturation concentrations), provided by theQifikit. Cells and beads were stained for 45 min at 4° C. in the dark.The cells were washed once and all samples were resuspended in 100 μlFACS buffer. Samples were analyzed immediately on a multicolor flowcytometer and installed software (e.g. Cantoll device running FACS Divasoftware or FACSCalibur flow cytometer using the CellQUEST software).

TABLE 17 BCMA specific antigen binding capacity on patient bone marrowmyeloma plasma cells as measured by quantitative flow cytometry analysisPatient No SABC_(BCMA) P1 n/a P2 n/a P3 679 P4 145 P5 957 P6 969 P7 554P8 4479 P9 350 P10 414 P11 2756 P12 2911 P13 1267 P14 3453 P15 1006 P161097 P17 1622 P18 429 P19 1684 P20 383 P21 1602 P22 799 P23 204

Example 13: Redirected T-Cell Cytotoxicity of Bone Marrow PatientMyeloma Plasma Cells in Presence of Autologous Bone Marrow InfiltratingT Cells Induced by Anti-BCMA/Anti-CD3 T Cell Bispecific Antibodies(Multiparameter Flow Cytometry)

One of the most meaningful and critical in vitro characterization duringpreclinical evaluation of TCB antibody candidates for multiple myelomais whether the TCB molecule could activate the patients' T cells andinduce redirected T-cell killing of primary myeloma plasma cells fromthe patients' bone marrow. To evaluate the effect of anti-BCMA/anti-CD3TCB antibodies to induce redirected T-cell killing of bone marrowmyeloma plasma cells, whole bone marrow aspirates were collected frommultiple myeloma patients in EDTA-coated tubes and immediately used forthe cell culture assays. The ratio of effector cells to tumor cells (E:Tratio) present in the whole bone marrow samples was determined andmeasured by flow cytometry. Briefly, 200 μl of bone marrow samples weretransferred into 96 deep-well plates. Anti-BCMA/anti-CD3 TCB antibodyand control antibody dilutions were prepared in sterile medium and 10 μlof the preparation were added to the respective wells for finalconcentrations ranging from 0.1 pM to 30 nM. The bone marrow-antibodysuspension is mixed by gentle shaking and then incubated at 37° C., 5%CO₂ for 48 h, sealed with paraffin film. After the incubation period, 20μl of a corresponding FACS antibody solution prepared based on anantibody-panel includingCD138-APCC750/CD38-FITC/CD5-BV510/CD56-PE/CD19-PerCP-Cy7/CD45-V450/BCMA-APC/Annexin-V-PerCP-Cy5.5were added into a 96-U-bottom plate. Fluorochrome-labelled antibodieswere purchased from BD Biosciences (San Jose, Calif.) and CaltagLaboratories (San Francisco Calif.) and in-house APC-conjugatedanti-human BCMA antibody was used. The samples were then incubated for15 minutes in the dark at room temperature and acquired and analyzedusing a multicolor flow cytometer. Cell death of the myeloma cells wasdetermined by evaluating annexin-V positive expression gated on themyeloma cell populations CD138⁺ CD38⁺ CD45⁺ CD19⁻ CD56⁺. Percentage ofmyeloma cell death was then determined. The percentage of lysis ofpatient bone marrow myeloma plasma cells induced by a specificconcentration of anti-BCMA/anti-CD3 T cell bispecific antibody was alsodetermined by measuring the absolute count of annexin-V-negative myelomaplasma cells at a given TCB concentration and subtracting it from theabsolute count of annexin-V-negative myeloma plasma cells without TCB;divided by the absolute count of annexin-V-negative myeloma plasma cellswithout TCB. To verify the specificity of the anti-BCMA/anti-CD3 T cellbispecific antibodies, annexin-V expression was also measured in otherbone marrow cell types such as T cells, B cells and NK cells. As shownin FIG. 8, there was a concentration-dependent and specific lysis ofpatient myeloma plasma cells while lysis of T cells, B cells, and NKcells was not observed. In addition, control-TCB which binds to CD3 onlybut not to BCMA did not induce cell death of myeloma plasma cells at thehighest concentrations of TCB antibodies. As shown in Table 18,percentage of annexin-V positive patient bone marrow myeloma cells atthe highest concentration (30 nM) reached up to 52.54% and 55.72% for42-TCBcv and 22-TCBcv respectively as compared to 29.31% for83A10-TCBcv, concluding that 42-TCBcv and 22-TCBcv are more potent than83A10-TCBcv to induce killing of patient bone marrow myeloma plasmacells.

TABLE 18 Percentage of annexin-V positive myeloma plasma cells frompatient bone marrow aspirates induced by anti- BCMA/anti-CD3 T cellbispecific antibodies. Annexin-V positive Anti-BCMA/anti-CD3 T cellbispecific myeloma plasma antibody concentration (pM) cells (%) 3000010000 1000 100 10 0 83A10-TCBcv 29.31 30.95 23.14 15.74 16.76 13.1142-TCBcv 52.54 39.87 29.96 10.51 19.6 13.11 22-TCBcv 55.72 51.71 31.0114.81 14.19 13.11 Control-TCB 15.18 10.93 / / / /

In another study in bone marrow aspirates from 5 different MM patients,the percentage of viable myeloma plasma cells was determined by gatingon annexin-V negative cell population and plotted against theconcentration of anti-BCMA/anti-CD3 T cell bispecific antibody. The EC50values were measured and determined as the TCB antibody concentrationthat results in 50% of maximum viable myeloma plasma cells. EMAX (%) wasdetermined as maximum of viable myeloma plasma cells in presence ofrespective anti-BCMA/anti-CD3 T cell bispecific antibody. 83A10-TCBcvwas much less potent in inducing lysis of myeloma plasma cells than22-TCBcv and 42-TCBcv in majority of the five myeloma patient bonemarrow aspirate samples (Table 26; FIG. 9 shows as example concentrationresponse curves for 2 of the 5 patients). Concentration-dependentreduction of viable myeloma cells was observed in 5/5 patient samplestreated with 22-TCBcv or 42-TCBcv, as compared to only 1/5 patientsamples for 83A10-TCBcv. Table 19 shows the comparison of 83A10-TCBcvwith 22-TCBcv and 42-TCBcv and the effect of the anti-BCMA/anti-CD3 Tcell bispecific antibodies on viability of bone marrow myeloma plasmacells. The results clearly show that there were less viable bone marrowmyeloma plasma cells with 22-TCBcv and 42-TCBcv (i.e. more lysis of thebone marrow myeloma plasma cells) in 4/5 patient samples as demonstratedby lower EMAX (%) values for 22-TCBcv and 42-TCBcv vs. 83A10-TCBcv inrespective patient samples. Concentration-dependent and specific lysisof patient myeloma plasma cells were observed while lysis ofnon-malignant bone marrow cells was not observed (data not shown).

TABLE 19 EMAX (%) values in respect to annexin-V negative viable myelomaplasma cells from patient bone marrow aspirates in presence of byanti-BCMA/anti-CD3 T cell bispecific antibodies. Bone marrow aspirate83A10-TCBcv 22-TCBcv 42-TCBcv patient sample (Study 2) EMAX (%) Patient001 100 7.6 22.6 Patient 003 54.3 38.9 44.6 Patient 004 100 66.6 53.9Patient 006 81.8 65.9 73.5 Patient 007 81.8 48.6 72.8

In a further investigations of the new anti-BCMA/anti-CD3 T cellbispecific antibodies used in this invention compared to 83A10-TCBcv,seven freshly taken patient whole bone marrow samples/aspirates werestained with CD138 magnetic microbeads (Miltenyi Biotec, BergischGladbach, Germany), passed through an autoMACS cell separation columnand the collected fractions with sufficient remaining number of MMplasma cells of usually >4% myeloma plasma cells were used for furtherexperiments. In 24-well plates, 500,000 cells/well were incubated andcultured for 48 hours. Anti-BCMA/anti-CD3 TCB antibodies and controlantibody dilutions were added to the respective wells for a final TCBconcentration of 0.1. pM to 10 nM. Each dose point was done intriplicates. Viability of the plasma cells and cells of the bone marrowmicroenvironment was investigated by propidium iodide/CD138-FITCdouble-staining using flow cytometry (FACSCalibur; Becton Dickinson).Data analysis was performed using FACSDiva Software (Becton Dickinson).As depicted in FIG. 10, bar plots show mean values normalized on themean over the triplicates of the respective medium control (MC). Forstatistical analysis, a one-sided t-test was used. The maximuminhibition of MM plasma cell growth at a concentration of 10 nM (IMAX10)and the inhibition measured at 1 nM (IMAX1), respectively, were given inpercent as referred to the medium control. The maximum inhibition of thecontrol-TCB antibody (10 nM) compared to the medium control was alsodepicted. Computations were performed using R 3.1.19, and Bioconductor2.1310, but for calculation of the IMAX values (Microsoft Excel®;Microsoft Office Professional 2013). An effect was consideredstatistically significant if the P-value of its correspondingstatistical test was <5% (*), <1% (**) or <0.1% (***). As shown in FIGS.10A-10G, the results clearly show that there were less viable bonemarrow myeloma plasma cells with 22-TCBcv and 42-TCBcv (i.e. more lysisof the bone marrow myeloma plasma cells) in 7/7 patient samples ascompared to 83A10-TCBcv. Table 20 demonstrates the percentage of viablemyeloma plasma cells from patient bone marrow aspirates induced byanti-BCMA/anti-CD3 T cell bispecific antibodies relative to mediumcontrol. Table 21 shows the IMAX10 and IMAX1 values.

The results demonstrate that 22-TCBcv and 42-TCBcv are clearly morepotent than 83A10-TCBcv to induce killing of patient bone marrow myelomaplasma cells. Despite specific lysis of bone marrow plasma cells (BMPC)induced by the anti-BCMA/anti-CD3 T cell bispecific antibodies andobserved in all bone marrow patient samples, the bone marrowmicroenvironment (BMME) was unaffected in the respective samples (FIG.10H, representative of 7 patient samples).

TABLE 20 Relative percentage of propidium iodide negative viable myelomaplasma cells from patient bone marrow aspirates induced byanti-BCMA/anti-CD3 T cell bispecific antibodies. Anti-BCMA/anti-CD3 Tcell bispecific antibody concentration (nM) 0.01 0.1 1 10 Patient sampleNo. 1/Viable myeloma plasma cells (%) 83A10-TCBcv 181.3  106.3  31.3 9.442-TCVcv 81.3 15.6 9.4 9.4 22-TCVcv 37.5  6.3 6.3 9.4 Ctrl-TCB / / /162.5 Patient sample No. 2/Viable myeloma plasma cells (%) 83A10-TCBcv89.5 31.6 5.3 0 42-TCVcv 42.1 10.5 0 0 22-TCVcv 15.8  5.3 0 0 Ctrl-TCB // / 94.7 Patient sample No. 3/Viable myeloma plasma cells (%)83A10-TCBcv 76.7 35.0 1.7 0 42-TCVcv 13.3 0  0 0 22-TCVcv  3.3 0  0 0Ctrl-TCB / / / 86.7 Patient sample No. 4/Viable myeloma plasma cells (%)83A10-TCBcv 93.9 51.5 9.1 6.1 42-TCVcv  9.1 0  0 0 22-TCVcv 15.2 15.2 00 Ctrl-TCB / / / 127.3 Patient sample No. 5/Viable myeloma plasma cells(%) 83A10-TCBcv 100   91.4 62.9 20.0 42-TCVcv 71.4 34.3 22.9 11.422-TCVcv 20.0 22.9 14.3 11.4 Ctrl-TCB / / / 85.7 Patient sample No.6/Viable myeloma plasma cells (%) 83A10-TCBcv 55.6 22.2 6.7 4.4 42-TCVcv35.6  6.7 4.4 4.4 22-TCVcv 24.4  3.3 8.9 2.2 Ctrl-TCB / / / 117.8Patient sample No. 7/Viable myeloma plasma cells (%) 83A10-TCBcv 84.482.6 46.8 19.3 42-TCVcv 67.0 33.9 12.8 5.5 22-TCVcv 24.4  3.3 8.9 2.2Ctrl-TCB / / / 106.4

TABLE 21 IMAX10 and IMAX1 values in respect to maximal inhibition of MMplasma cell growth at 10 nM IMAX10 and inhibition at 1 nM IMAX1 based onpropidium iodide negative viable myeloma plasma cells from patient bonemarrow aspirates in presence of by anti-BCMA/anti-CD3 T cell bispecificantibodies. Patient 83A10-TCBcv 42-TCBcv 22-TCBcv Ctrl-TCB Sample IMAX10IMAX1 IMAX10 IMAX1 IMAX10 IMAX1 IMAX10 No. (%) (%) (%) (%) (%) (%) (%) 190.6 68.8 90.6 90.6 90.6 93.8 −62.5 3 100 94.7 100 100 100 100 5.3 4 10098.3 100 100 100 100 13.3 5 93.9 90.9 100 100 100 100 −27.3 6 80.0 37.188.6 77.1 88.6 85.7 14.3 7 95.6 93.3 95.6 95.6 97.8 91.1 −17.8 8 80.753.2 94.5 87.2 97.2 97.2 −6.4

Example 14: T-Cell Activation of Patient Bone Marrow T Cells Induced byAnti-BCMA/Anti-CD3 T Cell Bispecific Antibodies (Multiparameter FlowCytometry)

To evaluate whether anti-BCMA/anti-CD3 TCB antibodies induce activationof myeloma patient CD4⁺ and CD8⁺ T cells (i.e. bone marrow infiltrated Tcells (MILs)), the samples from the respective treated, untreated andcontrol groups after 48 h of incubation were also stain with a FACSantibody solution prepared based on an antibody-panel including eightmarkers: CD8/CD69/TIM-3/CD16/CD25/CD4/HLA-DR/PD-1. The samples were thenincubated for 15 minutes in the dark at room temperature and acquiredand analyzed using a multicolor flow cytometer. T-cell activation wasdetermined by evaluating CD25, CD69 and/or HLA-DR positive expressiongated on CD4⁺ and CD8⁺ T-cell populations. Percentages of T-cellactivation were then measured. FIG. 11 shows a concentration-dependentupregulation of CD69 and CD25 on bone marrow-infiltrated CD4⁺ and CD8⁺ Tcells from multiple myeloma patients. Table 22 summarizes the increaseof CD69 and CD25 expression on CD4⁺ and CD8⁺ T cells induced byanti-BCMA/anti-CD3 TCB antibodies; data from one patient.

TABLE 22 T-cell activation of myeloma patient autologous T cells inducedby anti-BCMA/anti-CD3 T-cell bispecific antibodies in presence ofpatient bone marrow myeloma plasma cells Anti-BCMA/anti-CD3 T cellbispecific antibody concentration (pM) 30000 10000 1000 100 10 0CD69+/CD4 T cells (%) 83A10-TCBcv 21.8 14.93 1.80 0.93 1.02 0.8542-TCBcv 29.6 24.8 1.90 1.57 0.94 0.85 22-TCBcv 34.99 30.72 3.62 1.692.31 0.85 Control-TCB 0.7 0.62 / / / / CD69+/CD8 T cells (%) 83A10-TCBcv25.50 22.07  8.330 5.60 5.14 5.30 42-TCBcv 23.61 24.22 11.125 9.26 6.285.30 22-TCBcv 25.48 28.14 11.460 6.64 14.08  5.30 Control-TCB 5.71 4.93/ / / / CD25+/CD4 T cells (%) 83A10-TCBcv 17.47 12.86 5.18 4.58 4.077.5  42-TCBcv 8.65 7.42 3.51 2.71 2.81 7.5  22-TCBcv 12.34 11.52 5.234.89 4.90 7.5  Control-TCB 6.90 6.50 / / / / CD25+/CD8 T cells (%)83A10-TCBcv 9.79 6.560 0.42 0.13 0.12 0.12 42-TCBcv 2.20 2.231 0.42 0.140.08 0.12 22-TCBcv 3.57 4.110 0.65 0.10 0.08 0.12 Control-TCB 0.09 0.100/ / / /

Example 15: Increased T-Cell Function (Cytokine Production) of PatientBone Marrow T Cells Induced by Anti-BCMA/Anti-CD3 T Cell BispecificAntibodies (Multiplexed-Bead Based Immunoassay/Flow Cytometry)

To evaluate whether anti-BCMA/anti-CD3 TCB antibodies (83A10-TCBcv,22-TCBcv and 42-TCBcv) induce T-cell activation and increased functionof myeloma patient bone marrow infiltrating CD4+ and CD8⁺ T cells,supernatant were collected from the culture of the respective treated,untreated and control groups after 48 h of incubation and the content ofcytokines and serine proteases were measured. The cytokine bead array(CBA) analysis is performed on a multicolor flow cytometer according tomanufacturer's instructions, using either the Human Th1/Th2 Cytokine KitII (BD #551809) or the combination of the following CBA Flex Sets: humangranzyme B (BD #560304), human IFN-γ Flex Set (BD #558269), human TNF-αFlex Set (BD #558273), human IL-10 Flex Set (BD #558274), human IL-6Flex Set (BD #558276), human IL-4 Flex Set (BD #558272), human IL-2 FlexSet (BD #558270).

Example 16: Pharmacokinetic/Pharmacodynamic (PK/PD) Study in CynomolgusMonkeys

A clear advantage an anti-BCMA/anti-CD3 TCBcv antibody could have overother bispecific antibodies such as (scFV)₂ (e.g. BCMA×CD3 bispecificT-cell engager BiTE® as described in WO2013072415 and WO2013072406) isthe much longer elimination half-life/lower clearance in vivo whichcould allow a twice or once a week IV or SC administration as comparedto the very short elimination half-life of (scFV)₂ (e.g. 1 to 4 hours)requiring treatment administered via a pump carried by the patients forweeks to months (Topp et al. J Clin Oncol 2011; 29(18): 2493-8). A twiceor once a week administration would be much more convenient for thepatients and also much less risky (e.g. failure of pump, issues with thecatheter, etc.).

a) To verify the elimination half-life/clearance of anti-BCMA/anti-CD383A10-TCBcv antibody in vivo, single dose pharmacokinetic (PK)pharmacodynamic (PD) studies with anti-BCMA/anti-CD3 T-cell bispecificantibodies (83A10-TCBcv, 22-TCBcv and 42-TCBcv) were conducted atexperienced AAALAC-accredited CRO. Biologically naïve adult cynomolgusmonkeys of about two years old and weighing approximately 3 kg wereacclimatized for at least 40 days and selected on the basis of bodyweight, clinical observations and clinical pathology examinations.Animals were identified by Individual tattoos and color-coded cagecards. All the animal procedures (including housing, health monitoring,restrain, dosing, etc) and ethical revision was performed according tothe current country legislation enforcing the Directive on theprotection of animals used for biomedical research. Animals wererandomly assigned to the treatment group based on the most recentpretest body weight. After excluding animals with unacceptable pretestfindings, a computer program included in the Pristima® system designedto achieve balance with respect to pretest body weights was used toexclude animals from both body weight extremes and randomize theremaining animals to the treatment group. Animals were assigned to threetreatment groups with 83A10-TCBcv (n=2 animals i.e. 1 female and 1 maleper group) at 0.003; 0.03; and 0.3 mg/kg. Animals received a single i.v.injection of 83A10-TCBcv and at least 0.8 mL of blood samples pertimepoint were collected via the peripheral vein for PK evaluationsaccording to the following collection schedule and procedures: Pre-dose,30, 90, 180 min, 7, 24, 48, 96, 168, 336, 504 h after dosing. Bloodsamples were allowed to clot in tubes for serum separation for 60 min atroom temperature. The clot was spun down by centrifugation (at least 10min., 1200 g, +4° C.). The resultant serum (about 300 μL) was directlystored at −80° C. until further analysis. Bone marrow samples for PKevaluations were also collected at the femur under anesthesia/analgesictreatment according to the following collection schedule: Pre-dose, 96and 336 h after dosing. Bone marrow samples were allowed to clot intubes for serum separation for 60 min at room temperature. The clot wasspun down by centrifugation (at least 10 min, 1200 g, +4° C.). Theresultant bone marrow (about 1 mL) was directly stored at −80° C. untilfurther analysis. The PK data analysis and evaluation are performed.Standard non compartmental analysis is performed using Watson package (v7.4, Thermo Fisher Scientific Waltman, Mass., USA) or Phoenix WinNonlinsystem (v. 6.3, Certara Company, USA). As shown in FIG. 12 and Table 23,serum concentrations of 83A10-TCBcv were measured by ELISA from serumsamples collected at different timepoints after IV injection. Table 24shows the concentrations of 83A10-TCBcv in bone marrow as measured byELISA for each treatment group (BLQ means below level ofquantification).

Several information relevant for potential clinical use of thebispecific antibody can be taken from FIG. 12, Table 23 and Table 24:

-   -   In bone marrow aspirates from MM patients, concentrations of 1        nM or 10 nM of TCBs used in this invention induce significant or        even total killing of MM plasma cells; at the dose 0.03 mg/kg in        the interval from injection to 168 hours (7 days) plasma        concentrations between approx. 1 nM and 4 nM have been achieved        showing that once a week therapy with doses of approx. 0.03        mg/kg may well be feasible (200 ng/ml corresponds to approx. 1        nM)    -   FIG. 12 shows that in the investigated dose range PK is largely        dose linear; that means concentrations are proportional to dose;        a useful property for clinical therapy    -   MM is a disease mainly located in the bone marrow;        Concentrations of 83A10-TCBcv detected in bone marrow are close        to serum concentrations (Table 24), e.g. at 96 h after injection        bone marrow concentrations of approx. 1 and 2 nM have been        measured; these are concentrations of TCB used in this invention        at which significant killing of MM plasma cells is observed in        bone marrow aspirates freshly taken from MM Patients;        demonstrating again the opportunity for convenient dosing        intervals like once a week    -   Between 24 and 504 hours post injection, the elimination is        largely first order with an elimination half-life of approx. 6        to 8 days showing again the opportunity for e.g. once a week        dosing

TABLE 23 Serum concentrations of 83A10-TCBcv after IV treatment incynomolgus monkeys 83A10-TCBcv 0.003 mg/ 0.03 mg/ 0.3 mg/ Conc. kg IV kgIV kg IV (ng/mL) A B C D E F Pre-dose 0.00 0.00 0.00 0.00 0.00 0.00 30min 75.69 74.99 668.66 796.54 17207.20 14943.95 90 min 70.92 74.56951.81 628.72 12831.54 16248.97 180 min 76.54 62.55 981.42 722.2710653.28 6824.72 7 h 53.17 77.39 700.67 972.38 8204.77 4560.36 24 h33.16 50.41 358.90 532.11 4609.28 4127.41 48 h 26.05 37.40 279.80 433.303546.09 2700.43 96 h 17.28 19.52 226.01 429.80 1959.96 2006.92 168 h17.33 15.87 55.58 365.67 1918.06 1382.57 336 h 11.21 4.43 102.94 153.541102.96 773.55 504 h 4.33 BLQ 43.99 130.14 952.03 377.04

TABLE 24 Bone marrow concentrations of 83A10-TCBcv after single IVtreatment in cynomolgus monkeys Conc. 0.003 mg/kg 0.03 mg/kg 0.3 mg/kg(ng/mL) A B C D E F Pre-dose 0.00 0.00 0.00 0.00 0.00 0.00  96 h 25.0737.15 179.87 469.08 3432.54 2674.70 336 h 9.92 6.90 59.39 47.22 1987.48850.87

Pharmacodynamics (PD) measurements: Blood samples (timepoints: pre-dose,24, 48, 96, 168, 336, 504 h after dosing) and bone marrow samples(timepoints: pre-dose, 96 and 336 hs after dosing) were collected intubes containing 7.5% K3 EDTA for PD evaluation by flow cytometry toevaluate the effect of 83A10-TCBcv give i.v. as single dose on blood andbone marrow plasma cells, B cells, and T cells. A “lyse and wash” directimmunofluorescence staining method of the surface markers was applied.Briefly, 100 μL of blood or bone marrow was incubated with two antibodymixtures including CD45/CD2/CD16/CD20/CD27/CD38 orCD45/CD2/CD16/CD4/CD25/CD8 in the dark for 30 min at +4° C. To lyse redblood cells, 2 mL of lysing buffer solution was added to the sample andincubated 15 min at room temperature in the dark. Cells were collectedby centrifugation and washed with staining buffer (PBS 2% Fetal BovineSerum). The stained samples were kept refrigerated, protected fromlight, until acquisition with cytometer on the same day. FACS dataacquisition was performed with a Becton Dickinson flow cytometerequipped with 488 and 635 laser lines, BD FACS Canto II. BD FACSDivasoftware was used for data collection and analysis. The absolute cellnumber enumeration was performed with a double platform, based upon theWBC count obtained by the hematology analyzer (ADVIA™ 120, Siemens). Asshown in FIG. 13, peripheral T-cell redistribution was observed in allanimals receiving a single dose IV treatment of 83A10-TCBcv as shown bythe decrease in circulating T cell counts. As shown in FIG. 14A, alreadyat 24 h after treatment with 83A10-TCBcv 0.3 mg/kg a decrease in bloodplasma cells (BCMA-positive cells) was observed in animals treated whilethere was no decrease in total B cells (BCMA-negative cells). FIG. 14bshows the kinetic of plasma cell reduction in blood after treatment with83A10-TCBcv 0.3 mg/kg in cynomolgus monkeys.

Blood samples were also processed for plasma collection for cytokineanalysis (IL-1b, IL-2, IL-6, IL-10, TNF-α and IFN-γ) in accordance withthe following collection schedule: Pre-dose, 30, 90, 180 min, 7, 24, 48,96, 168 h after dosing. Blood samples were put in plastic tubes kept inan ice-water bath, then centrifuged (at least 10 min., 1200 g, +4° C.).The resultant plasma was directly stored at −80° C. until analysis.Cytokines analysis is performed with Multiplex bead-based cytokineimmunoassay (Luminex Technology).

Data are analyzed using Bio-Plex Manager 4.1 software (Bio-Rad): afive-parameter logistic regression model (5PL) is used.

b) In a further study, cynomolgus monkeys were treated with 42-TCBcv or22-TCBcv. Animals (n=2/group) received a single IV (0.01; 0.1; and 1.0mg/kg) or SC (0.01 and 0.1 mg/kg) injection of 42-TCBcv or single IVinjection with 22-TCBCv (0.1 mg/kg). Blood and bone marrow samples arecollected at timepoints following a defined collection schedule andprocessed accordingly for PK and PD measurement (immunophenotyping andcytokine production).

Animals received a single IV or SC. injection of 42-TCBcv or 22-TCBcv(only IV) and blood samples per timepoint were collected via theperipheral vein for PK evaluations according to the following collectionschedule and procedures: Pre-dose, 30, 90, 180 min, 7, 24, 48, 96, 168,336, 504 h after dosing. Blood samples were allowed to clot in tubes forserum separation for 60 min at room temperature. The clot was spun downby centrifugation (at least 10 min., 1200 g, +4° C.). The resultantserum (about 300 μL) was directly stored at −80° C. until furtheranalysis. Bone marrow samples for PK evaluations were also collected atthe femur under anesthesia/analgesic treatment according to thefollowing collection schedule: Pre-dose, 96 and 336 h after dosing. Bonemarrow samples were allowed to clot in tubes for serum separation for 60min at room temperature. The clot was spun down by centrifugation (atleast 10 min, 1200 g, +4° C.). The resultant bone marrow (about 1 mL)was directly stored at −80° C. until further analysis. The PK dataanalysis and evaluation were performed. Standard non compartmentalanalysis was performed using Watson package (v 7.4, Thermo FisherScientific Waltman, Mass., USA) or Phoenix WinNonlin system (v. 6.3,Certara Company, USA). As shown in FIG. 19 and Table 24A-D,concentrations of 42-TCBcv were measured by ELISA from serum and bonemarrow samples collected at different timepoints after IV or SCinjection. Effective concentration range of 42-TCBcv in multiple myelomapatient bone marrow aspirates corresponding to 10 μm to 10 nM (greyarea). Concentrations in parenthesis are in nM. BLQ, below level ofquantification; i/m, inconclusive measurement.

TABLE 24A Serum concentrations of 42-TCBcv after IV treatment incynomolgus monkeys 42-TCBcv Conc. 0.01 mg/kg IV 0.1 mg/kg IV 1.0 mg/kgIV (ng/mL) Male Female Male Female Male Female Pre-dose BLQ BLQ i/m BLQBLQ BLQ 30 min 468.57 613.44 4720.33 4506.64 41939.31 32677.23 90 min333.09 427.16 4284.66 3214.61 30889.73 103925.73 180 min 392.37 422.364336.89 2865.36 29201.69 36157.78 7 h 421.96 356.34 4028.47 3070.8425064.81 29962.62 24 h 242.64 305.74 2996.24 2321.66 19365.86 23656.6548 h i/m 192.97 2595.62 1781.91 20539.59 13523.68 96 h 128.50 148.022153.34 1277.02 13147.09 12755.58 168 h 51.13 72.64 1388.24 948.316189.79 3952.05 336 h 27.68 13.03 195.51 190.87 5337.85 54.15 504 h18.17 8.04 275.93 13.96 3678.69 37.88

TABLE 24B Bone marrow concentrations of 42-TCBcv after single IVtreatment in cynomolgus monkeys 42-TCBcv Conc. 0.01 mg/kg IV 0.1 mg/kgIV 1.0 mg/kg IV (ng/mL) Female Male Female Female Male Female Pre-doseBLQ BLQ 406.99 BLQ BLQ BLQ  96 h 54.39 130.03 956.56 1022.87 4089.884339.33 336 h 27.23 18.49 227.20 170.34 3705.74 62.44

TABLE 24C Serum concentrations of 42-TCBcv after SC treatment incynomolgus monkeys 42-TCBcv Conc. 0.01 mg/kg SC 0.1 mg/kg SC (ng/mL)Male Female Male Female Pre-dose 4.76 12.41 BLQ BLQ 30 min 8.25 12.5125.11 14.62 90 min 16.38 22.71 140.73 145.39 180 min 23.75 48.51 334.95269.66 7 h 37.46 63.48 836.86 565.10 24 h 68.15 115.31 2100.42 904.22 48h 116.63 118.03 1956.60 1111.06 96 h 150.77 120.62 1810.13 1817.52 168 h106.28 98.64 1192.65 1653.26 336 h 67.02 46.21 482.39 571.04 504 h 25.6931.99 4.08 83.91

TABLE 24D Bone marrow concentrations of 42-TCBcv after single SCtreatment in cynomolgus monkeys 42-TCBcv Conc. 0.01 mg/kg SC 0.1 mg/kgSC (ng/mL) Female Male Female Female Pre-dose 5.59 10.70 BLQ BLQ  96 h109.88 73.93 1064.66 1066.79 336 h 29.35 48.78 518.40 906.48

The results from Tables 24A and 24C show an attractive serumconcentration profile suitable for once a week or even once every twoweeks treatment with 42-TCBcv. Area under the curve AUC for serumconcentrations after IV and SC administration were determined,comparison of the AUC values showed high bioavailability of close to100% with SC injection of 42-TCBcv. In addition, the results show thatconcentration of 42-TCBcv in bone marrow is very similar to 42-TCBcvserum concentrations. 42-TCBcv concentrations in the serum could wellrepresent the concentrations of 42-TCBcv available in the bone marrowi.e. at the main location where the myeloma tumor cells are enriched.

Pharmacodynamic (PD) measurements are valuable information tocorroborate with PK measurements. Further PD analyses were performed.Cynomolgus CD20⁺ B cells from blood also express BCMA on the cellsurface and are significantly more frequent (higher absolute count) thanplasma cells in blood. Blood B-cell depletion was used as a reliablepharmacodynamic effect of anti-BCMA/anti-CD3 TCBcv antibodies and tocompare the in vivo efficacy between 83A10-TCBcv, 42-TCBcv and 22-TCBcv.Absolute B-cell counts were calculated based on the double platformconsisting of flow cytometry and WBC count obtained with a hematologyanalyser and measured at the following timepoints: pre-dose, 24h, 48h,96h and 196h after 10-min IV infusion. The percentage of B-celldepletion was calculated as followed:

$= \frac{\begin{matrix}{\left\lbrack {{absolute}\mspace{14mu} B\text{-}{cell}\mspace{14mu}{count}\mspace{14mu}{at}\mspace{14mu}{pre}\text{-}{dose}} \right\rbrack -} \\\left\lbrack {{absolute}\mspace{14mu} B\text{-}{cell}\mspace{14mu}{count}\mspace{14mu}{at}\mspace{14mu}{timepoint}} \right\rbrack\end{matrix}}{\left\lbrack {{absolute}\mspace{14mu} B\text{-}{cell}\mspace{14mu}{count}\mspace{14mu}{at}\mspace{14mu}{pre}\text{-}{dose}} \right\rbrack*100}$

TABLE 24E Pharmacodynamic effects of anti-BCMA/anti- CD3 TCBcvantibodies: B-cell depletion B-cell depletion relative to pre-dose (%)Time after 83A10-TCBcv 42-TCBcv 22-TCBcv IV injection 0.3 mg/kg 0.1mg/kg 0.1 mg/kg (hours) (n = 2) (n = 2) (n = 2) 24 h 19.9 ± 0.21 91.4 ±3.8 77.8 ± 3.7 48 h 11.9 ± 17.6 88.8 ± 3.9 61.5 ± 9.8 96 h  5.0 ± 10.893.0 ± 7.2 89.2 ± 4.8 168 h  −0.23 ± 61.4  96.6 ± 3.5 91.9 ± 3.9

42-TCBcv and 22-TCBcv are more potent than 83A10-TCBcv to inducedepletion of BCMA-expressing B cells in cynomolgus monkeys following asingle dose IV injection (see Table 24E). Since the three moleculesshare the same molecular structure and CD3 binder, the difference inefficacy in cynomolgus monkeys could be mainly attributed to therespective BCMA antibody.

To confirm that depletion of BCMA-expressing B cells in cynomolgusmonkeys after IV injection is a result of the mechanisticpharmacodynamic effects of anti-BCMA/anti-CD3 TCBcv antibodies, theincrease of activated CD8⁺ cytotoxic T cells (i.e. effector cells) wasmeasured in the bone marrow enriched of BCMA-positive cells (i.e. targetcells) 4 days (96 h) and 3 weeks (336h) after IV injection. AbsoluteCD8⁺ CD25⁺ activated T-cell counts were calculated based on the doubleplatform consisting of flow cytometry and WBC count obtained with ahematology analyser

TABLE 24F Pharmacodynamic effects of anti-BCMA/anti-CD3 TCBcvantibodies: Increase in CD8⁺ CD25⁺ activated T cells Increase in CD8⁺CD25⁺ activated T cells relative to pre-dose (%) Time after 83A10-TCBcv42-TCBcv 22-TCBcv IV injection 0.3 mg/kg 0.1 mg/kg 0.1 mg/kg (hours) (n= 2) (n = 2) (n = 2)  96 h  284 ± 244% 585 ± 496% 1449 ± 1715% 336 h−0.9 ± 1.3% 110 ± 187% −6.6 ± 45.3%

42-TCBcv and 22-TCBcv are more potent than 83A10-TCBcv to induce T-cellactivation in cynomolgus monkeys following a single dose IV injection(see Table 24F). Since the three molecules share the same molecularstructure and CD3 binder, the difference in pharmacodynamic effects incynomolgus monkeys could be mainly attributed to the respective BCMAantibody. The results indicate that depletion of BCMA-positive B cellsin bone marrow and in blood is most likely the result of activation ofcytotoxic T cells induced by anti-BCMA/anti-CD3 TCBcv antibodies.

Example 17: Antitumoral Activity Induced by Anti-BCMA/Anti-CD3 T CellBispecific Antibody in the H929 Human Myeloma Xenograft Model UsingPBMC-Humanized NOG Mice

With a long elimination half-life, Fc-containing anti-BCMA/anti-CD3TCBcv antibodies could be more efficacious than (scFv)₂-based bispecificantibodies such as BCMA50-BiTE® given at equimolar doses, in a once aweek schedule. The in vivo effect of 83A10-TCBcv and BCMA50-BiTE® (asdescribed in WO2013072415 and WO2013072406) was compared and evaluatedin the H929 human myeloma xenograft model in PBMC-humanized NOG mice.NOG mice are appropriate for humanized mouse models as they completelylack of immune cells including resident NK cell population and aretherefore more permissive to tumor engraftment of human xenogeneic cells(Ito et al. Curr Top Microbiol Immunol 2008; 324: 53-76). Briefly, onday 0 (d0) of the study, 5×10⁶ human myeloma cell line NCI-H929(NCI-H929, ATCC® CRL-9068™) in 100 μL RPMI 1640 medium containing 50:50matrigel (BD Biosciences, France) were subcutaneously (SC) injected intothe right dorsal flank of immunodeficient NOD/Shi-scid IL2rgamma(null)(NOG) female mice of 8-10 weeks of age (Taconic, Ry, Danemark).Twenty-four to 72 hours prior to H929 tumor cell SC implantation, allmice received a whole body irradiation with a 7-source (1.44 Gy, ⁶⁰Co,BioMep, Breteniéres, France). On day 15 (d15), NOG mice received asingle intraperitoneal (IP) injection of 2×10⁷ human PBMCs (in 500 μLPBS 1× pH7.4). Characterization of the human PBMC was performed byimmunophenotyping (flow cytometry). Mice were then carefully randomizedinto the different treatment and control groups (n=9/group) using VivoManager® software (Biosystemes, Couternon, France) and a statisticaltest (analysis of variance) was performed to test for homogeneitybetween groups. Antibody treatment started on day 19 (d19), i.e. 19 daysafter SC injection of H929 tumor cells when the tumor volume had reachedat least 100-150 mm³ in all mice, with a mean tumor volume of 300±161mm³ for the vehicle treated control group, 315±148 mm³ for the 2.6 nM/kgcontrol-TCB treated group, 293±135 mm³ for the 2.6 nM/kg 83A10-TCBcvgroup and 307±138 mm³ for the 2.6 nM/kg BCMA50-(scFv)₂ (BCMA50-BiTE®)group. The TCB antibody treatment schedule was based on thepharmacokinetic results previously obtained with 83A10-TCBcv andconsisted of a once a week IV administration for up to 3 weeks (i.e.total of 3 injections of TCB antibody). Four days after reconstitutionof the host mice with human PBMCs (d19), a first dose of theanti-BCMA/anti-CD3 83A10-TCBcv antibody (2.6 nM/kg respectively 0.5mg/kg) was given via tail vein injection. Blood samples were collectedby jugular/mandibular vein puncture (under anesthesia) 1 h before eachtreatment, 2 h before the second treatment and at termination in micefrom all groups treated with 83A10-TCBcv and control-TCBcv. Bloodsamples were immediately transferred into clot activator containingtubes (T MG tubes, cherry red top, Capiject®, Terumo®). Tubes were leftat room temperature for 30 min to allow clotting. Then tubes werecentrifuged at 1,300 g for 5 min for clot/serum separation. Serumaliquots were prepared, flash frozen in liquid nitrogen and stored at−80° C. until further analysis. Tumor volume (TV) was measured bycaliper during the study and progress evaluated by intergroup comparisonof TV. The percentage of tumor growth defined as TG (%) was determinedby calculating TG (%)=100× (median TV of analysed group)/(median TV ofcontrol vehicle treated group). For ethical reason, mice were euthanizedwhen TV reached at least 2000 mm³. FIG. 15 shows the TV of eachindividual mouse per experimental group: (A) control groups includingvehicle control (full line) and control-TCB (dotted line), (B)83A10-TCBcv (2.6 nM/kg) group, and (C) BCMA50-BiTE® (2.6 nM/kg). In the83A10-TCBcv (2.6 nM/kg) group, 6 out of 9 mice (67%) had their tumorregressed even below TV recorded at d19 i.e. first TCB treatment andtumor regression was maintained until termination of study. The 3 micein the 83A10-TCBcv (2.6 nM/kg) treated group which failed to show tumorregression had their TV equal to 376, 402 and 522 mm³ respectively atd19. In contrast, none of the 9 mice (0%) treated with an equimolar doseof BCMA50-BITE® (2.6 nM/kg) at a once a week schedule for 3 weeks hadtheir tumor regressed at any timepoints. Table 25 shows progression oftumor volumes over time in all experimental groups. The percentage oftumor growth was calculated for d19 to d43 and compared between83A10-TCBcv (2.6 nM/kg) group and BCMA50-BiTE® (2.6 nM/kg) (FIG. 16).The results demonstrate that TG (%) is consistently and significantlyreduced in the 83A10-TCBcv (2.6 nM/kg) group as well as the TG (%) isalways lower when compared to BCMA50-BiTE® (2.6 nM/kg). Table 26 showsthe median tumor volume (TV) and percentage of tumor growth (TG (%)) atdays 19 to 43. The overall results clearly demonstrated that 83A10-TCBcvis superior to BCMA50-BiTE® to induce antitumor activity in vivo whentreatment is given at equimolar dose in once a week schedule for 3weeks.

TABLE 25 Progression of tumor volumes over time in mice from controlvehicle group and mice treated with equimolar doses of control-TCB,83A10-TCBcv and BCMA50-(scFv)₂ (BCMA50-BiTE ®) Tumor volume Controlvehicle Group A (mm³) A1 A2 A3 A4 A5 A6 A7 A8 A9 Mean SD Day 5 95 58 6371 63 68 67 65 36 65 15 Day 8 70 61 71 70 56 68 74 70 49 66 8 Day 12 6665 53 50 57 58 60 59 56 58 5 Day 15 101 95 131 80 61 65 89 37 161 91 37Day 19 333 327 566 123 197 191 444 92 427 300 161 Day 23 565 481 1105470 310 309 517 281 581 513 249 Day 27 1071 877 1989 823 560 675 1089530 870 943 440 Day 30 1870 1129 x 419.2 867 1060 1368 673 1331 1090 450Day 34 x 1653 507 1056 1521 1805 1008 2042 1370 535 Day 37 2140 20431309 2017 2394 1267 x 1862 464 Day 40 x x 1592 x x 1346 1469 174 Day 431548 1994 1771 314 Day 47 x x Day 51 Tumor volume 2.6 nM/kg Control TCBGroup B (mm³) B1 B2 B3 B4 B5 B6 B7 B8 B9 Mean SD Day 5 68 65 84 83 46 6373 74 67 69 11 Day 8 55 64 54 73 60 103 56 55 76 66 16 Day 12 45 92 7376 83 78 103 69 76 77 16 Day 15 72 169 64 99 69 150 223 115 88 117 54Day 19 257 334 71 318 268 460 602 236 285 315 148 Day 23 430 773 95 444553 738 808 381 461 520 227 Day 27 924 1252 232 780 768 1009 915 606 630791 289 Day 30 1191 1714 326 867 1230 1349 1118 817 783 1044 398 Day 341684 x 592 1466 1660 1954 1765 1180 576 1359 529 Day 37 2522 597 17351105 x x 1402 861 1370 691 Day 40 x 978 2388 1952 2277 1365 1792 604 Day43 1302 x x x 1895 1599 419 Day 47 2346 2373 2359 19 Day 51 x x Tumorvolume 2.6 nM/kg 83A10-TCBcv Group C (mm³) C1 C2 C3 C4 C5 C6 C7 C8 C9Mean SD Day 5 78 79 55 77 53 47 39 53 60 60 15 Day 8 69 37 67 75 62 5959 77 75 64 12 Day 12 58 61 60 69 48 59 46 63 87 61 12 Day 15 136 41 61138 48 57 76 71 217 94 58 Day 19 376 151 238 522 154 133 377 287 402 293135 Day 23 656 322 375 847 311 249 642 395 681 498 210 Day 27 1119 376443 1400 253 253 678 371 1166 673 441 Day 30 1607 187 260 1975 88 113219 191 1590 692 783 Day 34 2143 68 100 x 34 54 63 53 2429 618 1033 Day37 x 41 44 43 34 34 35 x 38 5 Day 40 64 40 43 38 32 39 43 11 Day 43 4043 33 24 32 25 33 8 Day 47 14 21 16 12 19 14 16 3 Day 51 15 30 20 20 1518 20 6 Tumor volume 2.6 nM/kg BCMA50-(scFv)₂ (BCMA50-BiTE ®) Group D(mm³) D1 D2 D3 D4 D5 D6 D7 D8 D9 Mean SD Day 5 75 92 78 86 57 91 74 5862 75 13 Day 8 51 87 61 99 70 88 90 73 71 77 15 Day 12 70 73 63 76 84 7685 58 113 78 16 Day 15 142 72 61 128 87 77 121 60 188 104 44 Day 19 232212 81 474 303 260 360 304 539 307 138 Day 23 560 483 121 811 665 408654 457 1115 586 278 Day 27 827 879 216 1224 1092 732 886 908 1526 921359 Day 30 1026 1414 227 1476 1373 1256 1210 1228 2433 1294 567 Day 341368 1855 418 2185 1734 1936 1465 1645 x 1576 535 Day 37 1691 2754 5992542 2102 2062 1958 765 Day 40 2764 x 706 x x x 1735 1455 Day 43 x 807807 n/a Day 47 x Day 51

TABLE 26 Median tumor volume (TV) and percentage of tumor growth (TG(%)) at days 19 to 43: 83A10-TCBcv in comparison to BCMA50-BiTE ®. TumorVehicle treated 83A10-TCBcv BCMA50-BiTE ® Control-TCB growth Control 2.6nM/kg 2.6 nM/kg 2.6 nM/kg inhibition Median TG Median TG Median TGMedian TG TG_(inh) (%) TV (%) TV (%) TV (%) TV (%) Day 19 327 100 28787.8 303 92.7 285 87.2 Day 23 481 100 395 82.1 560 116.4 461 95.8 Day 27870 100 443 50.9 886 101.8 780 89.7 Day 30 1094.5 100 219 20.0 1256114.8 1118 102.1 Day 34 1521 100 65.5 4.3 1689.5 111.1 1563 102.8 Day 372030 100 38 1.9 2082 102.6 1253.5 61.7 Day 40 1469 100 39.5 2.7 1735118.1 1952 132.9 Day 43 1771 100 32.5 1.8 807 45.6 1598.5 90.3 Day 47 // 15 / / / 2359.5 / Day 51 / / 19 / / / / /

Example 19: Redirected T-Cell Cytotoxicity of Plasma Cells fromPeripheral Blood Mononuclear Cells or Bone Marrow Aspirates of Patientwith Plasma Cell Leukemia (PCL) in Presence of Autologous T CellsInduced by Anti-BCMA/Anti-CD3 T Cell Bispecific Antibodies as Measuredby Flow Cytometry

Plasma cell leukemia (PCL) is a leukemic variant of myeloma arisingeither de novo or from clinically pre-existent multiple myeloma (MM).The current available treatments are rather limited and consist mainlyof combinations of MM drugs and chemotherapy. To date no therapy hasever been explicitly registered for this highly aggressive and deadlydisease. BCMA plays an essential role in the survival of normal plasmacells and an anti-BCMA/anti-CD3 T cell bispecific antibodies can be usedfor plasma cell leukemia treatment in a patient suffering from saiddisease. Freshly taken peripheral blood mononuclear cells (PBMC) fromplasma cell leukemia patient samples containing >80% plasma cells athigh leucocyte counts are isolated by density gradient using Ficoll orother comparable methods and incubated for 24h and 48h withanti-BCMA/anti-CD3 T cell bispecific antibody concentrations or controlantibodies of 0.1 pM to 30 nM at 37° C. in a humidified air atmosphere.Whole bone marrow aspirates from plasma cell leukemia patients can alsobe used as samples. Each dose point is done in triplicates. Apoptosis isdetermined by annexin/propidium iodide staining of the whole populationand of the CD138 positive cells on a FACSCalibur using Diva software(BD). Viability of the plasma cells and PBMC whole population areinvestigated by propidium iodide/CD138-FITC double-staining using flowcytometry (FACSCalibur; Becton Dickinson). Data analysis is performedusing FACSDiva Software (Becton Dickinson). Mean values are normalizedon the mean over the triplicates of the respective medium control (MC).For statistical analysis, a one-sided t-test is used. The maximuminhibition of PCL cell growth at a concentration of 10 nM (IMAX10) andthe inhibition measured at 1 nM (IMAX1), respectively, are given inpercent as referred to the medium control. The maximum inhibition of thecontrol-TCB antibody (10 or 30 nM) compared to the medium control isalso measured. Computations are performed using R 3.1.19, andBioconductor 2.1310, but for calculation of the IMAX values (MicrosoftExcel®; Microsoft Office Professional 2013). An effect is consideredstatistically significant if the P-value of its correspondingstatistical test is <5% (*), <1% (**) or <0.1% (***). BCMA expression isalso measured on PBMC CD138⁺ plasma cells from plasma cell leukemiapatient samples as well as effector cells to tumor cells (E:T) ratio isdetermined. As shown in FIG. 20, the results clearly show that there wassignificantly reduced viable bone marrow plasma cell leukemic cells with42-TCBcv (i.e. more lysis of the bone marrow plasma cell leukemic cells)in two plasma cell leukemia patient samples as compared to the mediumcontrol Table 27 demonstrates the percentage of maximum inhibition ofplasma cell leukemic cells from patient bone marrow aspirates orperipheral blood induced by 10 nM (IMAX10) and 1 nM (IMAX1)anti-BCMA/anti-CD3 T cell bispecific antibodies relative to mediumcontrol. The results demonstrate that 42-TCBcv is very potent to inducekilling of patient bone marrow plasma cell leukemic cells. Despitespecific lysis of bone marrow plasma cell leukemic cells induced by theanti-BCMA/anti-CD3 T cell bispecific antibodies and observed bone marrowsamples (PCL patient 1), the bone marrow microenvironment (BMME) wasunaffected in the respective samples (data not shown).

The killing of the malignant plasma cells in patients with PCL by usingEM901 is potent. But at least in one of the two patient samples testedat the highest concentration of 10 nM killing is only a little bit above50%. Experiments with the combinations of EM901 with either thalidomidesor PD-1 or PD-L1 antibodies or with anti CD38 antibodies will show thepotential benefit of such combinations, e.g. by higher % of killing ate.g. 10 nM or other concentrations of EM901

TABLE 27 IMAX10 and IMAX1 values in respect to maximal inhibition ofplasma cell leukemia plasma cell growth at 10 nM (IMAX10) and inhibitionat 1 nM (IMAX1) based on propidium iodide negative viable plasma cellleukemic cells from patient bone marrow aspirates in presence of byanti-BCMA/anti-CD3 T cell bispecific antibodies. 42-TCBcv Ctrl-TCBPatient IMAX10 IMAX1 IMAX10 Sample No. (%) (%) (%) 1 99.6 88.2 −2.7 2~60.0 ~40.0 ~8.0

Example 20: Redirected T-Cell Cytotoxicity of Bone Marrow Plasma Cellsfrom Patient with AL Amyloidosis in Presence of Autologous T CellsInduced by Anti-BCMA/Anti-CD3 T Cell Bispecific Antibodies as Measuredby Flow Cytometry

AL amyloidosis is a rare disease caused by a disorder of the bone marrowwhich usually affects people from ages 50-80 and with two-third of thepatients being male. AL amyloidosis is reflected by an abnormalproduction of antibody/immunoglobulin protein by the plasma cells. In ALamyloidosis, the light chains (LC) of the antibody are misfolded and theabnormal LC misfolded protein result is the formation of amyloid. Thesemisfolded amyloid proteins are deposited in and around tissues, nervesand organs. As the amyloid builds up in an organ, nerve or tissue, itgradually causes damage and affects their function. Patients with ALamyloidosis are often affected with more than one organ. Since BCMAplays an essential role in the survival of normal plasma cells, it ishighly justified to evaluate the effect of anti-BCMA/anti-CD3 T cellbispecific antibodies in killing plasma cells in AL amyloidosis. Freshlytaken AL amyloidosis patient whole bone marrow samples/aspirates areeither exposed directly to the anti-BCMA/anti-CD3 TCB antibodies orstained with CD138 magnetic microbeads (Miltenyi Biotec, BergischGladbach, Germany), passed through an autoMACS cell separation columnand the collected fractions with sufficient remaining number of ALamyloidosis plasma cells of usually >4% are used for furtherexperiments. In 24-well plates, 500,000 cells/well are incubated andcultured for 48 hours. Anti-BCMA/anti-CD3 TCB antibodies and controlantibody dilutions are added to the respective wells for a final TCBconcentration of 0.1 pM to 30 nM. Each dose point is done intriplicates. Viability of the plasma cells and cells of the bone marrowmicroenvironment is investigated by propidium iodide/CD138-FITCdouble-staining using flow cytometry (FACSCalibur; Becton Dickinson).Data analysis is performed using FACSDiva Software (Becton Dickinson).In addition also combinations of EM901 with thalidomides or anti-PD-1antibodies or anti-PD-L1 antibodies or with anti-CD38 antibodies aretested in the same experimental set-up. Various concentrations of EM901as well as of the combination partners will be tested. Mean values arenormalized on the mean over the triplicates of the respective mediumcontrol (MC). For statistical analysis, a one-sided t-test is used. Themaximum inhibition of PCL cell growth at a concentration of 10 nM(IMAX10) and the inhibition measured at 1 nM (IMAX1), respectively, aregiven in percent as referred to the medium control. The maximuminhibition of the control-TCB antibody (10 or 30 nM) compared to themedium control is also measured. The same values are also determinedfrom the results obtained in combinations, e.g. maximum inhibition ate.g. 10 nM EM9091 in the presence of x nM of e.g. lenalidomide etc.Computations are performed using R 3.1.19, and Bioconductor 2.1310, butfor calculation of the IMAX values (Microsoft Excel®; Microsoft OfficeProfessional 2013). An effect is considered statistically significant ifthe P-value of its corresponding statistical test is <5% (*), <1% (**)or <0.1% (***). BCMA expression is also measured on bone marrow CD138⁺plasma cells from AL amyloidosis patient samples as well as effectorcells to tumor cells (E:T) ratio is determined.

Example 21: Combined Use of Anti-BCMA/Anti-CD3 T Cell BispecificAntibody with an Immunotherapeutic Drug

Anti-BCMA/anti-CD3 T-cell bispecific antibody (namely 83A10-TCBcv or42-TCBcv) was combined with three drugs representing different types ofimmunotherapy: lenalidomide, anti-PD-1 antibody and anti-CD38 antibodydaratumumab. H929 MM cells were co-cultured with human leukocytes fromhealthy donors (n=1 or 5) in RPMI-1640 medium supplemented with 10% FBS,0.5% gentamincin and 1% L-Glutamine at 37° C. in a humidified 5% CO₂incubator and challenged to suboptimal concentrations of 83A10-TCBcv (10pM) (n=5) or 42-TCBcv (10 pM) (n=1) alone, or in combination withlenalidomide (1 μM), anti-PD-1 antibody (10 μg/ml) and anti-CD38antibody daratumumab (10 μg/ml). As shown in FIG. 21A, combining83A10-TCBcv with lenalidomide or daratumumab significantly increasedtheir anti-MM efficacy by 4-fold and 2.5-fold, respectively. FIG. 21Band Table 28 show the percentage of MM cell lysis when H929 cells andleucocytes from one healthy donor were exposed to 42-TCBcv (10 pM)combined with lenalidomide (1 μM), anti-PD-1 antibody (10 μg/ml) andanti-CD38 antibody daratumumab (10 μg/ml) for 48 h of culture. Combining42-TCBcv with lenalidomide or daratumumab also increased their anti-MMefficacy to kill MM cell lines.

TABLE 28 MM cell lysis with anti-BCMA/anti-CD3 TCB alone or combinedwith lenalidomide, anti-PD-1 antibody or anti-CD38 antibody daratumumab:example with donor 1. Donor 1 42-TCBcv alone (10 pM) 6.74% Combinationof 42-TCBcv: without TCB with TCB Daratumumab (10 μg/ml) 33.06% 42.29%lenalidomide (1 μM) −1.78% 13.27% Anti-PD-1 20.26% 23.56%

Example 22: Combined In Vivo Use of Anti-BCMA/Anti-CD3 T Cell BispecificAntibody with an Anti-PD-1 or Anti-PD-L1 Antibody

The additive or synergistic combination of anti-BCMA/anti-CD3 TCB withan antibody to block PD-1/PD-L1 pathway (either with an anti-PD-1antibody or anti-PD-L1 antibody) and inhibit the down-modulation ofT-cell responses upon engagement of PD-L1 with its receptor PD-1 on Tcells can be optimally observed in an in vivo setting such as a MMxenograft model or MM mouse model instead of in vitro. Briefly, on day 0(d0) of the study, 5×10⁶ human myeloma cell line NCI-H929 (NCI-H929,ATCC® CRL-9068™) in 100 μL RPMI 1640 medium containing 50:50 matrigel(BD Biosciences) are subcutaneously (SC) injected into the right dorsalflank of immunodeficient NOD/Shi-scid IL2rgamma(null) (NOG) female miceof 8-10 weeks of age (Taconic, Ry, Danemark). On day 15 (d15), NOG micereceive a single intraperitoneal (IP) injection of 2×10⁷ human PBMCs (in500 μL PBS 1× pH7.4). Characterization of the human PBMC is performed byimmunophenotyping (flow cytometry). Mice are then carefully randomizedinto the different treatment and control groups (n=9/group) and astatistical test (analysis of variance) is performed to test forhomogeneity between groups. Antibody treatment starts on day 19 (d19),i.e. 19 days after SC injection of H929 tumor cells when the tumorvolume reaches at least 100-150 mm³ in all mice. The TCB antibodytreatment schedule is based on the pharmacokinetic results previouslyobtained with anti-BCMA/anti-CD3 TCB and consisted of a once a week IVadministration for up to 3 weeks (i.e. total of 3 injections of TCBantibody). Two to four days after reconstitution of the host mice withhuman PBMCs (d19), a first weekly dose of the anti-BCMA/anti-CD3antibody (dose 0.1 to 0.5 mg/kg) is given via tail vein injection aloneor in combination with an anti-PD-L1 antibody also given once/week (dose1 to 10 mg/kg). Tumor volume (TV) is measured by caliper during thestudy and progress is evaluated by intergroup comparison of TV. Thepercentage of tumor growth defined as TG (%) is determined bycalculating TG (%)=100× (median TV of analysed group)/(median TV ofcontrol vehicle treated group)

1.-10. (canceled)
 11. A method of treating multiple myeloma comprisingadministering to a patient in need of such treatment a therapeuticallyeffective amount of a) a bispecific antibody comprising a first bindingpart specifically binding to human B cell maturation antigen (BCMA) anda second binding part specifically binding to human CDR (CD3), whereinsaid first binding part comprises: a VH region comprising a CDR1H regionof SEQ ID NO:21, a CDR2H region of SEQ ID NO:22 and a CDR3H region ofSEQ ID NO:17 and a VL region comprising a CDR1L region of SEQ ID NO:27,a CDR2L region of SEQ ID NO:28 and a CDR3L region of SEQ ID NO:20; and,b) an immunotherapeutic drug selected from the group consisting ofthalidomide or an immunotherapeutic derivative thereof, an anti-CD38antibody, an anti-PD-1 antibody and an anti-PD-L1 antibody; wherein thebispecific antibody and the immunotherapeutic drug are administeredseparately or together.
 12. The method according to claim 11 wherein theimmunotherapeutic drug is selected from the group consisting ofdaratumumab, isatuximab, MOR202, Ab79, Ab19, thalidomide, lenalidomide,pomalidomide, CC-122, CC-220, pembrolizumab, pidilizumab, nivolumab,MEDI-0680, PDR001, REGN2810, lambrolizumab, MDX-1106, BGB-108, h409A11,h409A16, h409A17, atezolizumab, avelumab, durvalumab, and MDX-1105. 13.The method according to claim 12 wherein the immunotherapeutic drug isdaratumumab.
 14. The method according to claim 12 wherein theimmunotherapeutic drug is isatuximab.
 15. The method according to claim12 wherein the immunotherapeutic drug is MOR202.
 16. The methodaccording to claim 12 wherein the immunotherapeutic drug is Ab79. 17.The method according to claim 12 wherein the immunotherapeutic drug isAb19.
 18. The method according to claim 12 wherein the immunotherapeuticdrug is thalidomide.
 19. The method according to claim 12 wherein theimmunotherapeutic drug is lenalidomide.
 20. The method according toclaim 12 wherein the immunotherapeutic drug is pomalidomide.
 21. Themethod according to claim 12 wherein the immunotherapeutic drug isCC-122.
 22. The method according to claim 12 wherein theimmunotherapeutic drug is CC-220.
 23. The method according to claim 12wherein the immunotherapeutic drug is pembrolizumab.
 24. The methodaccording to claim 12 wherein the immunotherapeutic drug is pidilizumab.25. The method according to claim 12 wherein the immunotherapeutic drugis nivolumab.
 26. The method according to claim 12 wherein theimmunotherapeutic drug is MEDI-0680.
 27. The method according to claim12 wherein the immunotherapeutic drug is PDR001.
 28. The methodaccording to claim 12 wherein the immunotherapeutic drug is REGN2810.29. The method according to claim 12 wherein the immunotherapeutic drugis lambrolizumab.
 30. The method according to claim 12 wherein theimmunotherapeutic drug is MDX-1106.
 31. The method according to claim 12wherein the immunotherapeutic drug is BGB-108.
 32. The method accordingto claim 12 wherein the immunotherapeutic drug is h409A11.
 33. Themethod according to claim 12 wherein the immunotherapeutic drug ish409A16.
 34. The method according to claim 12 wherein theimmunotherapeutic drug is h409A17.
 35. The method according to claim 12wherein the immunotherapeutic drug is atezolizumab.
 36. The methodaccording to claim 12 wherein the immunotherapeutic drug is avelumab.37. The method according to claim 12 wherein the immunotherapeutic drugis durvalumab.
 38. The method according to claim 12 wherein theimmunotherapeutic drug is MDX-1105.